One possibility is that GIMAP6 is a passive cargo that GABARAPL2 escorts for degradation via the autophagolysosomal pathway in particular circumstances of tension (e.g. Amount S3: A 15kDa proteins Triciribine phosphate (NSC-280594) co-purifies with biotinylated GIMAP6 from transfected HEK293Tcells transiently. HEK293T cells had been transiently transfected using a plasmid encoding individual GIMAP6 in pcDNA3Biot1His6iresBirA or using the matching vector as indicated. Lysates were prepared 48 h later as well as the associated and biotinylated protein purified using streptavidin-agarose. The purified proteins had been separated by SDS Web page as well as the gel silver-stained. The electrophoretic mobilities from the purified GIMAP6 and an linked 15kDa proteins are indicated. The full total result shown is representative of two independent experiments.(TIF) pone.0077782.s003.tif (116K) GUID:?29D09F07-3E76-4CC1-AEEE-30D59FA6BF1A Amount S4: Endogenous GIMAP6 and GABARAPL2 neglect to co-immunoprecipitate. Cell lysates had been ready from Jurkat-T cells which acquired either been still left neglected or starved for 2 h as defined in the Components and Strategies section. Lysates had been after that either analysed straight by SDS-PAGE and Traditional western blotting for appearance of GIMAP6 and GABARAPL2 (-panel A left hands aspect) or MAP1LC3B (-panel B) or had been initial immunoprecipitated with rabbit anti individual GIMAP6 polyclonal antiserum (I) or the matching pre-immune serum (P) ahead of SDS Web page and Traditional western blotting (-panel A right hands aspect).(TIF) pone.0077782.s004.tif (170K) GUID:?F77B0029-6F61-4D78-B717-2CCA9DC9747C Amount S5: Sequence alignment of GIMAP6 protein sequences from several mammalian species. Proteins sequences had been either taken straight from the NCBI proteins database or had been deduced from portrayed DNA series tags or genomic sequences. The conserved AIG1/GTPase domains is normally boxed in dark and the expanded C-terminal regions within most mammals, but absent Felypressin Acetate from mouse, rat and chinese language hamster, boxed in crimson.(TIF) pone.0077782.s005.tif (1.0M) GUID:?BABAA9C9-3DB5-4097-99DD-A3F5BB704EE3 Figure S6: Mouse GIMAP6 associates weakly with GABARAPL2. HEK293T cells had been transfected with myc-GIMAP6-encoding plasmids with or without GABARAPL2 in pcDNA3Biot1His6iresBirA, as indicated. Lysates had been prepared, and associated and biotinylated protein purified in the lysates using streptavidin-agarose. Western blots from the retrieved protein had been probed with HRP-conjugated streptavidin (showing GABARAPL2) or a mouse monoclonal antibody (9E10) towards the myc-tag accompanied by an HRP-conjugated goat anti-mouse IgG (showing GIMAP6). Traditional western blots were developed using Immobilon ECL western blotting substrate. Interpretation of this experiment was complex as mouse GIMAP6 indicated only weakly in our transient assays compared with the human being orthologue. However, co-immunoprecipitation of mouse GIMAP6 with GABARAPL2 was shown in two self-employed experiments.(TIF) pone.0077782.s006.tif (141K) GUID:?22D296F4-57E5-4388-A941-FC4BDF87131D Number S7: The mouse anti-myc mAb 9E10 and a rabbit anti-human GIMAP6 polyclonal antiserum display related intracellular distribution of myc-GIMAP6. Myc-GIMAP6 HEK293 cells were either starved for 90 moments or remaining untreated and were then processed for immunocytochemistry, using main antibodies as indicated. The level bars show 16 m. The results demonstrated are representative of three self-employed experiments.(TIF) pone.0077782.s007.tif (1.4M) GUID:?A9147ED7-481E-4BEA-B2F4-B92D271BF132 Number S8: GIMAP6 over-expression does not affect MAP1LC3B-II accumulation. Cell lines (panel A C Biot-GIMAP6-His myc-BirA-Jurkat cell collection or the related parental cell collection; panel B C myc GIMAP6 T-Rex HeLa cell collection plus or minus tetracycline treatment; panel C C myc-GIMAP6 HEK293 cells or the related vector control cells) were starved for 2 h (panels A and B) or 1.5h (panel C) or remaining untreated, with or without treatment with chloroquine as indicated. Cell lysates were prepared and analysed by SDS PAGE and Western blotting with antibodies to MAP1LC3B and ACTIN. Resulting X-ray films were scanned and images analysed using ImageJ software to determine MAP1LC3B-II/ACTIN ratios. Panel D C analysis of MAP1LC3B-II/ACTIN ratios for three experiments within the cells from Panel A. Results are demonstrated as mean SD (n=3). The presence of chloroquine is definitely indicated by CQ. One-way ANOVA indicated no significant statistical variations between treatments in the absence or presence of GIMAP6.(TIF) pone.0077782.s008.tif (1.0M) GUID:?546555CA-1010-49C7-9D07-BFE615F66132 Figure S9: GIMAP6 expression does not affect the number of MAP1LC3 or SQSTM1 puncta/cell. myc-GIMAP6 HEK293 cells and the related vector cells were either starved for 90 min or remaining untreated and consequently immunocytochemically stained for MAP1LC3B or SQSTM1 as indicated. Standard images are demonstrated in panel A. The level bars represent 21 m. Places were counted (150-200 cells analysed for MAP1LC3B and 100-150 for SQSTM1 per condition). Analysis was performed using Imaris software. Results are offered as places/cell SD C panel B MAP1LC3B; panel C SQSTM1..The first possibility we considered was that GIMAP6 might be affecting the levels of mRNA. protein co-purifies with biotinylated GIMAP6 from transiently transfected HEK293Tcells. HEK293T cells were transiently transfected having a plasmid encoding human being GIMAP6 in pcDNA3Biot1His6iresBirA or with the related vector as indicated. Lysates were prepared 48 h later on and the biotinylated and connected proteins purified using streptavidin-agarose. The purified proteins were separated by SDS PAGE and the gel silver-stained. The electrophoretic mobilities of the purified GIMAP6 and an connected 15kDa protein are indicated. The result demonstrated is representative of two self-employed experiments.(TIF) pone.0077782.s003.tif (116K) GUID:?29D09F07-3E76-4CC1-AEEE-30D59FA6BF1A Number S4: Endogenous GIMAP6 and GABARAPL2 fail to co-immunoprecipitate. Cell lysates were prepared from Jurkat-T cells which experienced either been remaining untreated or starved for 2 h as explained in the Materials and Methods section. Lysates were then either analysed directly by SDS-PAGE and Western blotting for manifestation of GIMAP6 and GABARAPL2 (panel A left hand part) or MAP1LC3B (panel B) or were 1st immunoprecipitated with rabbit anti human being GIMAP6 polyclonal antiserum (I) or the related pre-immune serum (P) prior to SDS PAGE and Western blotting (panel A right hand side).(TIF) pone.0077782.s004.tif (170K) GUID:?F77B0029-6F61-4D78-B717-2CCA9DC9747C Physique S5: Sequence alignment of GIMAP6 protein sequences from various mammalian species. Protein sequences were either taken directly from the NCBI protein database or were deduced from expressed DNA sequence tags or genomic sequences. The conserved AIG1/GTPase domain name is usually boxed in black and the extended C-terminal regions present in most mammals, but absent from mouse, rat and chinese hamster, boxed in red.(TIF) pone.0077782.s005.tif (1.0M) GUID:?BABAA9C9-3DB5-4097-99DD-A3F5BB704EE3 Figure S6: Mouse GIMAP6 associates weakly with GABARAPL2. HEK293T cells were transfected with myc-GIMAP6-encoding plasmids with or without GABARAPL2 in pcDNA3Biot1His6iresBirA, as indicated. Lysates were prepared, and biotinylated and associated proteins purified from the lysates using streptavidin-agarose. Western blots of the recovered proteins were probed with HRP-conjugated streptavidin (to show GABARAPL2) or a mouse monoclonal antibody (9E10) to the myc-tag followed by an HRP-conjugated goat anti-mouse IgG (to show GIMAP6). Western blots were developed using Immobilon ECL western blotting substrate. Interpretation of this experiment was complex as mouse GIMAP6 expressed only weakly in our transient assays compared with the human orthologue. However, co-immunoprecipitation of mouse GIMAP6 with GABARAPL2 was exhibited in two impartial experiments.(TIF) pone.0077782.s006.tif (141K) GUID:?22D296F4-57E5-4388-A941-FC4BDF87131D Physique S7: The mouse anti-myc mAb 9E10 and a rabbit anti-human GIMAP6 polyclonal antiserum show comparable intracellular distribution of myc-GIMAP6. Myc-GIMAP6 HEK293 cells were either starved for 90 minutes or left untreated and were then processed for immunocytochemistry, using primary antibodies as indicated. The scale bars indicate 16 m. The results shown are representative of three impartial experiments.(TIF) pone.0077782.s007.tif (1.4M) GUID:?A9147ED7-481E-4BEA-B2F4-B92D271BF132 Physique S8: GIMAP6 over-expression does not affect MAP1LC3B-II accumulation. Cell lines (panel A C Biot-GIMAP6-His myc-BirA-Jurkat cell line or the corresponding parental cell line; panel B C myc GIMAP6 T-Rex HeLa cell line plus or minus tetracycline treatment; panel C C myc-GIMAP6 HEK293 cells or the corresponding vector control cells) were starved for 2 h (panels A and B) or 1.5h (panel C) or left untreated, with or without treatment with chloroquine as indicated. Cell lysates were prepared and analysed by SDS PAGE and Western blotting with antibodies to MAP1LC3B and ACTIN. Triciribine phosphate (NSC-280594) Resulting X-ray films were scanned and images analysed using ImageJ software to determine MAP1LC3B-II/ACTIN ratios. Panel D C analysis of MAP1LC3B-II/ACTIN ratios for three experiments around the cells from Panel A. Results are shown as mean SD (n=3). The presence of chloroquine is usually indicated by CQ. One-way ANOVA indicated no significant statistical differences between treatments in the absence or presence of GIMAP6.(TIF) pone.0077782.s008.tif (1.0M) GUID:?546555CA-1010-49C7-9D07-BFE615F66132 Figure S9: GIMAP6 expression does not Triciribine phosphate (NSC-280594) affect the number of MAP1LC3 or SQSTM1 puncta/cell. myc-GIMAP6 HEK293 cells and the corresponding vector cells were either starved for 90 min or left untreated and subsequently immunocytochemically stained for MAP1LC3B or SQSTM1 as indicated. Common images are shown in panel A. The scale bars represent 21 m. Spots were counted (150-200 cells analysed for MAP1LC3B and 100-150 for SQSTM1 per condition). Analysis was performed using Imaris software. Results are presented as spots/cell SD C panel B MAP1LC3B; panel C SQSTM1. No factor was observed in the true amount of places in the lack or existence of GIMAP6. Immunocytochemical results demonstrated are representative of three 3rd party tests.(TIF) pone.0077782.s009.tif (1.6M) GUID:?5B93E962-ADC3-4D14-BF62-08167A780B89 Abstract The GIMAPs (GTPases from the immunity-associated proteins) certainly are a family of little GTPases expressed prominently in the immune system systems of mammals and additional vertebrates. In mammals, research of mutant or genetically-modified rodents possess indicated important tasks for the GIMAP GTPases in the advancement and success of lymphocytes. No very clear picture has however emerged, however, from the molecular systems where they perform their function(s). Using biotin tag-affinity purification we determined a significant, and.As opposed to removing the 1st 10 proteins from the protein, mutation of the residue to alanine, which prevents the occurrence of proteolysis/lipidation, didn’t avoid the interaction of GABARAPL2 with GIMAP6 (Figure 3D), suggesting how the interaction may appear in the cytosol before GABARAPL2 becomes membrane-associated. GIMAP6 regulates the cellular GABARAPL2 level Throughout our studies, we pointed out that GIMAP6 over-expression could be affecting intracellular degrees of GABARAPL2. as well as the biotinylated and connected protein purified using streptavidin-agarose. The purified proteins had been separated by SDS Web page as well as the gel silver-stained. The electrophoretic mobilities from the purified GIMAP6 and an connected 15kDa proteins are indicated. The effect demonstrated is consultant of two 3rd party tests.(TIF) pone.0077782.s003.tif (116K) GUID:?29D09F07-3E76-4CC1-AEEE-30D59FA6BF1A Shape S4: Endogenous GIMAP6 and GABARAPL2 neglect to co-immunoprecipitate. Cell lysates had been ready from Jurkat-T cells which got either been remaining neglected or starved for 2 h as referred to in the Components and Strategies section. Lysates had been after that either analysed straight by SDS-PAGE and Traditional western blotting for manifestation of GIMAP6 and GABARAPL2 (-panel A left hands part) or MAP1LC3B (-panel B) or had been 1st immunoprecipitated with rabbit anti human being GIMAP6 polyclonal antiserum (I) or the related pre-immune serum (P) ahead of SDS Web page and Traditional western blotting (-panel A right hands part).(TIF) pone.0077782.s004.tif (170K) GUID:?F77B0029-6F61-4D78-B717-2CCA9DC9747C Shape S5: Sequence alignment of GIMAP6 protein sequences from different mammalian species. Proteins sequences had been either taken straight from the NCBI proteins database or had been deduced from indicated DNA series tags or genomic sequences. The conserved AIG1/GTPase site can be boxed in dark as well as the prolonged C-terminal regions within most mammals, but absent from mouse, rat and chinese language hamster, boxed in reddish colored.(TIF) pone.0077782.s005.tif (1.0M) GUID:?BABAA9C9-3DB5-4097-99DD-A3F5BB704EE3 Figure S6: Mouse GIMAP6 associates weakly with GABARAPL2. HEK293T cells had been transfected with myc-GIMAP6-encoding plasmids with or without GABARAPL2 in pcDNA3Biot1His6iresBirA, as indicated. Lysates had been ready, and biotinylated and connected proteins purified through the lysates using streptavidin-agarose. Traditional western blots from the retrieved proteins had been probed with HRP-conjugated streptavidin (showing GABARAPL2) or a mouse monoclonal antibody (9E10) towards the myc-tag accompanied by an HRP-conjugated goat anti-mouse IgG (showing GIMAP6). Traditional western blots had been created using Immobilon ECL traditional western blotting substrate. Interpretation of the experiment was complicated as mouse GIMAP6 indicated only weakly inside our transient assays weighed against the human being orthologue. Nevertheless, co-immunoprecipitation of mouse GIMAP6 with GABARAPL2 was proven in two 3rd party tests.(TIF) pone.0077782.s006.tif (141K) GUID:?22D296F4-57E5-4388-A941-FC4BDF87131D Shape S7: The mouse anti-myc mAb 9E10 and a rabbit anti-human GIMAP6 polyclonal antiserum display identical intracellular distribution of myc-GIMAP6. Myc-GIMAP6 HEK293 cells had been either starved for 90 a few minutes or left neglected and had been then prepared for immunocytochemistry, using principal antibodies as indicated. The range bars suggest 16 m. The outcomes proven are representative of three unbiased tests.(TIF) pone.0077782.s007.tif (1.4M) GUID:?A9147ED7-481E-4BEA-B2F4-B92D271BF132 Amount S8: GIMAP6 over-expression will not affect MAP1LC3B-II accumulation. Cell lines (-panel A C Biot-GIMAP6-His myc-BirA-Jurkat cell series or the matching parental cell series; -panel B C myc GIMAP6 T-Rex HeLa cell series plus or minus tetracycline treatment; -panel C C myc-GIMAP6 HEK293 cells or the matching vector control cells) had been starved for 2 h (sections A and B) or 1.5h (-panel C) or still left neglected, with or with no treatment with chloroquine as indicated. Cell lysates had been ready and analysed by SDS Web page and Traditional western blotting with antibodies to MAP1LC3B and ACTIN. Causing X-ray films had been scanned and pictures analysed using ImageJ software program to determine MAP1LC3B-II/ACTIN ratios. -panel D C evaluation of MAP1LC3B-II/ACTIN ratios for three tests over the cells from -panel A. Email address details are proven as mean SD (n=3). The current presence of chloroquine is normally indicated by CQ. One-way ANOVA indicated no significant statistical distinctions between remedies in the lack or existence of GIMAP6.(TIF) pone.0077782.s008.tif (1.0M) GUID:?546555CA-1010-49C7-9D07-BFE615F66132 Figure S9: GIMAP6 expression will not affect the amount of MAP1LC3 or SQSTM1 puncta/cell. Triciribine phosphate (NSC-280594) myc-GIMAP6 HEK293 cells as well as the matching vector cells had been either starved for 90 min or still left untreated and eventually immunocytochemically stained for MAP1LC3B or SQSTM1 as indicated. Usual images are proven in -panel A. The range pubs represent 21 m. Areas had been counted (150-200 cells analysed for MAP1LC3B and 100-150 for SQSTM1 per condition). Evaluation was performed using Imaris software program. Outcomes.Immunocytochemical analysis from the distribution of GIMAP6 in the HUVEC cells showed a cytoplasmic localisation in the control cells (Figure 6B(ii)). as well as the gel silver-stained. The electrophoretic mobilities from the purified GIMAP6 and an linked 15kDa proteins are indicated. The effect proven is consultant of two unbiased tests.(TIF) pone.0077782.s003.tif (116K) GUID:?29D09F07-3E76-4CC1-AEEE-30D59FA6BF1A Amount S4: Endogenous GIMAP6 and GABARAPL2 neglect to co-immunoprecipitate. Cell lysates had been ready from Jurkat-T cells which acquired either been still left neglected or starved for 2 h as defined in the Components and Strategies section. Lysates had been after that either analysed straight by SDS-PAGE and Traditional western blotting for appearance of GIMAP6 and GABARAPL2 (-panel A left hands aspect) or MAP1LC3B (-panel B) or had been initial immunoprecipitated with rabbit anti individual GIMAP6 polyclonal antiserum (I) or the matching pre-immune serum (P) ahead of SDS Web page and Traditional western blotting (-panel A right hands aspect).(TIF) pone.0077782.s004.tif (170K) GUID:?F77B0029-6F61-4D78-B717-2CCA9DC9747C Amount S5: Sequence alignment of GIMAP6 protein sequences from several mammalian species. Proteins sequences had been either taken straight from the NCBI proteins database or had been deduced from portrayed DNA series tags or genomic sequences. The conserved AIG1/GTPase domains is normally boxed in dark as well as the expanded C-terminal regions within most mammals, but absent from mouse, rat and chinese language hamster, boxed in crimson.(TIF) pone.0077782.s005.tif (1.0M) GUID:?BABAA9C9-3DB5-4097-99DD-A3F5BB704EE3 Figure S6: Mouse GIMAP6 associates weakly with GABARAPL2. HEK293T cells had been transfected with myc-GIMAP6-encoding plasmids with or without GABARAPL2 in pcDNA3Biot1His6iresBirA, as indicated. Lysates had been ready, and biotinylated and linked proteins purified in the lysates using streptavidin-agarose. Traditional western blots from the retrieved proteins had been probed with HRP-conjugated streptavidin (showing GABARAPL2) or a mouse monoclonal antibody (9E10) towards the myc-tag accompanied by an HRP-conjugated goat anti-mouse IgG (showing GIMAP6). Traditional western blots had been created using Immobilon ECL traditional western blotting substrate. Interpretation of the experiment was complicated as mouse GIMAP6 portrayed only weakly inside our transient assays weighed against the individual orthologue. Nevertheless, co-immunoprecipitation of mouse GIMAP6 with GABARAPL2 was confirmed in two indie tests.(TIF) pone.0077782.s006.tif (141K) GUID:?22D296F4-57E5-4388-A941-FC4BDF87131D Body S7: The mouse anti-myc mAb 9E10 and a rabbit anti-human GIMAP6 polyclonal antiserum present equivalent intracellular distribution of myc-GIMAP6. Myc-GIMAP6 HEK293 cells had been either starved for 90 mins or left neglected and had been then prepared for immunocytochemistry, using major antibodies as indicated. The size bars reveal 16 m. The outcomes proven are representative of three indie tests.(TIF) pone.0077782.s007.tif (1.4M) GUID:?A9147ED7-481E-4BEA-B2F4-B92D271BF132 Body S8: GIMAP6 over-expression will not affect MAP1LC3B-II accumulation. Cell lines (-panel A C Biot-GIMAP6-His myc-BirA-Jurkat cell range or the matching parental cell range; -panel B C myc GIMAP6 T-Rex HeLa cell range plus or minus tetracycline treatment; -panel C C myc-GIMAP6 HEK293 cells or the matching vector control cells) had been starved for 2 h (sections A and B) or 1.5h (-panel C) or still left neglected, with or with no treatment with chloroquine as indicated. Cell lysates had been ready and analysed by SDS Web page and Traditional western blotting with antibodies to MAP1LC3B and ACTIN. Ensuing X-ray films had been scanned and pictures analysed using ImageJ software program to determine MAP1LC3B-II/ACTIN ratios. -panel D C evaluation of MAP1LC3B-II/ACTIN ratios for three tests in the cells from -panel A. Email address details are proven as mean SD (n=3). The current presence of chloroquine is certainly indicated by CQ. One-way ANOVA indicated no significant statistical distinctions between remedies in the lack or existence of GIMAP6.(TIF) pone.0077782.s008.tif (1.0M) GUID:?546555CA-1010-49C7-9D07-BFE615F66132 Figure S9: GIMAP6 expression will not affect the amount of MAP1LC3 or SQSTM1 puncta/cell. myc-GIMAP6 HEK293 cells as well as the matching vector cells had been either starved for 90 min or still left untreated and eventually immunocytochemically stained for MAP1LC3B or SQSTM1 as indicated. Regular images are proven in -panel A. The size pubs represent 21 m. Areas had been counted (150-200 cells analysed for MAP1LC3B and 100-150 for SQSTM1 per condition). Evaluation was performed using Imaris software program. Results are shown as areas/cell SD C -panel B MAP1LC3B; -panel C SQSTM1. No factor was observed in the amount of areas in the lack or existence of GIMAP6. Immunocytochemical outcomes proven are representative of three indie tests.(TIF) pone.0077782.s009.tif (1.6M) GUID:?5B93E962-ADC3-4D14-BF62-08167A780B89 Abstract The GIMAPs (GTPases from the immunity-associated proteins) certainly are a family of little GTPases expressed prominently in the immune system systems of mammals and various other vertebrates. In mammals, research of.The suspensions were rotated at 4C for 4 h, and centrifuged at 15 then,000 g for 20 s. Body S3: A 15kDa proteins co-purifies with biotinylated GIMAP6 from transiently transfected HEK293Tcells. HEK293T cells had been transiently transfected using a plasmid encoding individual GIMAP6 in pcDNA3Biot1His6iresBirA or using the matching vector as indicated. Lysates had been ready 48 h afterwards and the biotinylated and associated proteins purified using streptavidin-agarose. The purified proteins were separated by SDS PAGE and the gel silver-stained. The electrophoretic mobilities of the purified GIMAP6 and an associated 15kDa protein are indicated. The result shown is representative of two independent experiments.(TIF) pone.0077782.s003.tif (116K) GUID:?29D09F07-3E76-4CC1-AEEE-30D59FA6BF1A Figure S4: Endogenous GIMAP6 and GABARAPL2 fail to co-immunoprecipitate. Cell lysates were prepared from Jurkat-T cells which had either been left untreated or starved for 2 h as described in the Materials and Methods section. Lysates were then either analysed directly by SDS-PAGE and Western blotting for expression of GIMAP6 and GABARAPL2 (panel A left hand side) or MAP1LC3B (panel B) or were first immunoprecipitated with rabbit anti human GIMAP6 polyclonal antiserum (I) or the corresponding pre-immune serum (P) prior to SDS PAGE and Western blotting (panel A right hand side).(TIF) pone.0077782.s004.tif (170K) GUID:?F77B0029-6F61-4D78-B717-2CCA9DC9747C Figure S5: Sequence alignment of GIMAP6 protein sequences from various mammalian species. Protein sequences were either taken directly from the NCBI protein database or were deduced from expressed DNA sequence tags or genomic sequences. The conserved AIG1/GTPase domain is boxed in black and the extended C-terminal regions present in most mammals, but absent from mouse, rat and chinese hamster, boxed in red.(TIF) pone.0077782.s005.tif (1.0M) GUID:?BABAA9C9-3DB5-4097-99DD-A3F5BB704EE3 Figure S6: Mouse GIMAP6 associates weakly with GABARAPL2. HEK293T cells were transfected with myc-GIMAP6-encoding plasmids with or without GABARAPL2 in pcDNA3Biot1His6iresBirA, as indicated. Lysates were prepared, and biotinylated and associated proteins purified from the lysates using streptavidin-agarose. Western blots of the recovered proteins were probed with HRP-conjugated streptavidin (to show GABARAPL2) or a mouse monoclonal antibody (9E10) to the myc-tag followed by an HRP-conjugated goat anti-mouse IgG (to show GIMAP6). Western blots were developed using Immobilon ECL western blotting substrate. Interpretation of this experiment was complex as mouse GIMAP6 expressed only weakly in our transient assays compared with the human orthologue. However, co-immunoprecipitation of mouse GIMAP6 with GABARAPL2 was demonstrated in two independent experiments.(TIF) pone.0077782.s006.tif (141K) GUID:?22D296F4-57E5-4388-A941-FC4BDF87131D Figure S7: The mouse anti-myc mAb 9E10 and a rabbit anti-human GIMAP6 polyclonal antiserum show similar intracellular distribution of myc-GIMAP6. Myc-GIMAP6 HEK293 cells were either starved for 90 minutes or left untreated and were then processed for immunocytochemistry, using primary antibodies as indicated. The scale bars indicate 16 m. The results shown are representative of three independent experiments.(TIF) pone.0077782.s007.tif (1.4M) GUID:?A9147ED7-481E-4BEA-B2F4-B92D271BF132 Figure S8: GIMAP6 over-expression does not affect MAP1LC3B-II accumulation. Cell lines (panel A C Biot-GIMAP6-His myc-BirA-Jurkat cell line or the corresponding parental cell line; panel B C myc GIMAP6 T-Rex HeLa cell line plus or minus tetracycline treatment; panel C C myc-GIMAP6 HEK293 cells or the related vector control cells) were starved for 2 h (panels A and B) or 1.5h (panel C) or remaining Triciribine phosphate (NSC-280594) untreated, with or without treatment with chloroquine as indicated. Cell lysates were prepared and analysed by SDS PAGE and Western blotting with antibodies to MAP1LC3B and ACTIN. Producing X-ray films were scanned and images analysed using ImageJ software to determine MAP1LC3B-II/ACTIN ratios. Panel D C analysis of MAP1LC3B-II/ACTIN ratios for three experiments within the cells from Panel A. Results are demonstrated as mean SD (n=3). The presence of chloroquine is definitely indicated by CQ. One-way ANOVA indicated no significant statistical variations between treatments in the absence or presence of GIMAP6.(TIF) pone.0077782.s008.tif (1.0M) GUID:?546555CA-1010-49C7-9D07-BFE615F66132 Figure S9: GIMAP6 expression does not affect the number of MAP1LC3 or SQSTM1 puncta/cell. myc-GIMAP6 HEK293 cells and the related vector cells were either starved for 90 min or remaining untreated and consequently immunocytochemically stained for MAP1LC3B or SQSTM1 as indicated. Standard images are demonstrated in panel A. The level bars represent 21 m. Places were counted (150-200 cells analysed for MAP1LC3B and 100-150 for SQSTM1 per condition). Analysis.