Fig. 3.
MCL1 is phosphorylated on serine 159 by GSK3β during mitophagy and GSK3β inhibition delays AMBRA1-dependent mitophagy. a HeLa cells were transfected with MYC-AMBRA1ActA or PcDNA3 and treated with the proteasome inhibitor MG132 (10 μM, 24 h) or not (DMSO). Total extracts were immunoblotted using the indicated antibodies and anti-HSP90 was used as a loading control. The graph illustrates the COXIV/HSP90 ratio (±S.D) as an arbitrary unit (a.u). Each point value represents the mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s test (****P < 0.0001, **P < 0.01). b HeLa cells were transfected with PcDNA3 or MYC-AMBRA1WT and treated with O/A (2.5 μM, 0.8 μM, 5 h) and proteasome inhibitor (MG132, 10 μM, 5 h) or not (DMSO). Total extracts were immunoblotted using the indicated antibodies and anti-HSP90 was used as a loading control. The graph illustrates the COXIV/HSP90 ratio (±S.D) as an aribitrary unit (a.u). Each point value represents the mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s test (*P < 0.05, **P < 0.01). c HeLa cells were transfected with MYC-AMBRA1ActA or PcDNA3 and treated with GSK3 inhibitor (SB-216763, 1 μM, 24 h) or not (DMSO). Total extracts were immunoblotted using the indicated antibodies and anti-HSP90 was used as a loading control. The graph illustrates the P-S159MCL1/MCL1 ratio (±S.D) as an arbitrary unit (a.u). Each point value represents the mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s test (*P < 0.05). d HeLa cells were transfected with PcDNA3 or MYC-AMBRA1WT and treated with O/A (2.5 μM, 0.8 μM, 5 h) and GSK3 inhibitor (SB-216763, 1 μM, 5 h) or not (DMSO). Total extracts were immunoblotted using the indicated antibodies and anti-HSP90 was used as a loading control. The graph illustrates the COXIV/HSP90 ratio (±S.D) as an arbitrary unit (a.u). Each point value represents the mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s test (*P < 0.05, **P < 0.01). e HeLa cells were cotransfected with MYC-AMBRA1ActA and PcDNA3 or GSK3β dead kinase (GSK3β-K85A). After 24 h of transfection, total extracts were immunoblotted using the indicated antibodies and anti-HSP90 was used as a loading control. The graph illustrates the COXIV/HSP90 ratio (±S.D) as an arbitrary unit (a.u). Each point value represents the mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s test (*P < 0.05). f HeLa cells were cotransfected with MYC-AMBRA1WT and PcDNA3 or GSK3β dead kinase (GSK3β-K85A) and treated with O/A (2.5 μM, 0.8 μM, 5 h). After 24 h of transfection, total extracts were immunoblotted using the indicated antibodies and anti-HSP90 was used as a loading control. The graph illustrates the COXIV/HSP90 ratio (±S.D) as an arbitrary unit (a.u). Each point value represents the mean ± S.D. from three independent experiments. Statistical analysis was performed using Student’s test (*P < 0.05)