Fig. 2: PJA1 promotes PGAM5 degradation by increasing its K48-linked ubiquitination.

a Silver staining of SDS–PAGE gels showed that the Flag-immunoprecipitants were pulled down from SUNE1 cells overexpressing Flag-PJA1. Red lines indicate the proteins of interest. b Co-IP with an anti-PJA1 antibody revealed the endogenous association of PJA1 and PGAM5 in NPC cells. c IF staining revealed the cellular localisation of exogenous Flag-PJA1 (purple), endogenous PGAM5 (green) and mitochondria (red) in NPC cells. Scale bars, 5 μm. d, e Protein (d) and mRNA (e, mean ± s.d., two-tailed unpaired t-test (left), one-way ANOVA with Dunnett’s multiple comparisons tests (right)) expression of PGAM5 in NPC cells transfected with gradient concentrations of the Flag-PJA1 plasmids, as well as in NPC cells transfected with the shCtrl or sh-PJA1s plasmids. f Immunoblot (left) and the corresponding greyscale analysis (right) of PGAM5 expression in HEK293T and HONE1 cells transfected with the HA-PGAM5 plasmids together with the empty vector or Flag-PJA1 plasmids after the CHX treatment (mean ± s.d., two-way ANOVA). g, h PGAM5 protein levels in NPC cells transfected with the empty vector or Flag-PJA1 plasmids after the treatment with MG132 (g) or CQ (h). i, j NPC cells transfected with the empty vector or Flag-PJA1 plasmids (i) or with the shCtrl or sh-PJA1 plasmids (j) together with Myc-PGAM5 and HA-WT-Ub or its mutants (HA-K48O-Ub, HA-K63O-Ub or HA-K48R-Ub) were subjected to denaturing IP with the indicated antibodies. n = 3 (e) independent experiments. Source data are provided as a Source Data file.