Fig. 4: PGAM5 dephosphorylates MST3.

a Immuno-purification and mass spectrometry analysis of HCT116 cells expressing flag-pcDNA3.1 or flag-MST3 on an anti-flag affinity column. b, c Co-IP assay of endogenous interaction between PGAM5 and MST3 in HCT116 cells after immunoprecipitation with either anti-PGAM5 antibody (b) or anti-MST3 antibody (c). d Western blotting for PGAM5, MST3 and pMST3^T178 in HCT116 cells transfected with flag-pcDNA3.1 or flag-PGAM5 plasmids. β-Actin was used as the loading control. e Western blotting for PGAM5, MST3 and pMST3^T178 proteins in HCT116 cells transfected with NC or siPGAM5. β-Actin was used as the loading control. f AlphaFold predicted binding fragment of MST3 to PGAM5. g Schematic diagram of construction of human PGAM5 phosphatase inactivation mutant plasmid (Histidine at position 105 changed to Alanine). h Western blotting for Flag, MST3 and pMST3^T178 proteins in HCT116 cells transfected with NC/siPGAM5 and then transfected with flag-PGAM5^WTor flag-PGAM5^H105A plasmids. β-Actin was used as the loading control. i Schematic diagram of the in vitro MST3 protein dephosphorylation assay. j Western blotting was used to evaluate the effect of flag-PGAM5^WT versus flag-PGAM5^H105A on MST3 phosphorylation described in panel (i). k Schematic representation of the sites where PGAM5 protein is cleaved. l Western blotting for Flag, MST3 and pMST3^T178 in LOVO cells transfected with PGAM5^WT or truncated PGAM5 (25-289). β-Actin was used as the loading control. m GST pull-down assays for interaction with GST-MST3 were performed in the presence or absence of His-SUMO-tagged PGAM5 (25-289). Samples were subjected to Western blotting. GST was used as the negative control. n Purified GST-tagged MST3 was combined with His-SUMO-tagged PGAM5 (25-289) to determine the amount of MST3 phosphorylation remaining by an in vitro dephosphorylation system. o Western blotting for Flag, MST3, pMST3^T178 in HCT116 cells with stable knockdown of PGAM5 (shPGAM5) transfected with flag-pcDNA3.1, flag-PGAM5^WT or flag-PGAM5^△23-29. β-Actin was used as the loading control. Western blotting images are representative of experiments that were repeated at least three times with similar results.