Fig 1: BAG3 promotes autophagy via upregulation of GLS.a Western blot analysis of changes induced by BAG3 overexpression in GLS, LC3 conversion and p62 levels in indicated cells. b–e HepG2 or MCF7 cells were infected with lentivirus containing empty or BAG3 construct, and glutamine consumption (b), intracellular glutamate production (c), intracellular a-KG (d), and ammonia production (e) were analyzed using the colorimetric method. f Control or BAG3-overexpressing HepG2 cells were transfected with scramble shRNA or shRNA specific against GLS (shGLS), and Western blot analysis was performed using the indicated antibodies. g and h Control or BAG3-overexpressing HepG2 cells were transfected with scramble shRNA or shRNA specific against GLS (shGLS), and glutamine consumption (g), ammonia production (h) were analyzed using the colorimetric method. i Control or BAG3-overexpressing MCF7 cells were transfected with scramble shRNA or shRNA specific against GLS (shGLS), and Western blot analysis was performed using the indicated antibodies. *P < 0.05. N.S. not significant. Error bars indicate means ± SD
Fig 2: Ectopic BAG3 expression increases GLS succinylation at Lys158 and Lys164 sites.a Globally screening succinylation proteomics identified that lysine 158 and lysine 164 are succinylated in succinylated GLS. b BAG3 overexpression leads to increased succinylation of GLS in HepG2 and MCF7 cells. c Immunoprecipitation assays of interaction between GLS and SIRT5 in HepG2 cells infected with lentivirus containing empty or BAG3 construct. Ectopic BAG3 expression decreases the interaction between GLS and SIRT5. d Western blot analysis of the levels of SIRT5 in control or BAG3-overexpressing cells. e Co-immunoprecipitation assays of interaction between BAG3 and GLS or SIRT5 in HepG2 cells
Fig 3: Succinylation at Lys158 and Lys164 sites stabilizes GLS via suppression its Lys48-linked ubiquitination.a–c HepG2 cells were infected with lentivirus containing empty, GLS (WT), GLS (K158/164 A) or GLS (K158/164E) construct. a Western blot analysis was performed using the indicated antibodies. Glutamine consumption (b) and ammonia production (c) were analyzed using the colorimetric method. d–f MCF7 cells were infected with lentivirus containing empty, GLS (WT), GLS (K158/164 A) or GLS (K158/164E) construct. d Western blot analysis was performed using the indicated antibodies. Glutamine consumption (e) and ammonia production (f) were analyzed using the colorimetric method. g and h The effect of overexpressing GLS (WT), GLS (K158/164 A) or GLS (K158/164E) on the half-life of GLS was investigated in indicated cells treated with cyclohexamide (CHX). i In vivo ubiquitination was performed by transfecting HepG2 or MCF7 cells with indicated expression plasmids. Cells were transfected with the indicated constructs for 36 h and incubated with MG132 for additional 4 h. Lysates were subjected to denaturing immunoprecipitation with an anti-Myc antibody and the levels of GLS (WT), GLS (K158/164 A) or GLS (K158/164E) ubiquitination were detected by immunoblotting with anti-HA antibody. j GLS polyubiquitination assay following transfection of HA-tagged ubiquitin (Ub) or its mutant forms K48 and K63. k Control or BAG3-overexpressing HepG2 cells were transfected with HA-Ub or its mutant forms K48 and K63, and analyzed by immunoprecipitation with an anti-Myc antibody and immunoblotting with the indicated antibodies. *P < 0.05. Error bars indicate means ± SD
Fig 4: BAG3 stabilizes GLS by suppression its proteasomal degradation. a HepG2 or MCF7 cells were infected with lentivirus containing empty or BAG3 construct, and GLS mRNA expression was measured using RT-qPCR. b The effect of overexpressing BAG3 on the half-life of GLS was evaluated in indicated cells treated with cyclohexamide (CHX). c Control or BAG3-overexpressing HepG2 and MCF7 cells were treated with vehicle, MG132 or E-64d plus pepstatin A respectively, GLS protein expression was investigated using Western blotting. *P < 0.05. N.S. not significant. Error bars indicate means ± SD
Supplier Page from MilliporeSigma for Monoclonal Anti-GLS antibody produced in mouse