Fig. 6
From: Functional role of PGAM5 multimeric assemblies and their polymerization into filaments
Cleaved PGAM5 forms oligomers in cells. a SDS-PAGE analysis of endogenous PGAM5 in HEK293T whole cell lysate (WCL), mitochondrial (mito), and cytoplasmic (cyto) fractions 4 h post-CCCP treatment. Uncleaved (UC) and cleaved (C) PGAM5 bands are marked by arrows. b BN-PAGE analysis of endogenous PGAM5 4 h post-CCCP treatment in mitochondrial (mito) and cytoplasmic (cyto) fractions. Fractionation samples were normalized based on the total protein concentrations determined for the WCL samples. As a reference for migration behavior of PGAM5 oligomers, recombinant ∆48 WT (∆48 WT) and the oligomerization-deficient mutant ∆48 F244E mutant (∆48 F244E) PGAM5 were run alongside cellular fractionation samples on a 4–16% native-PAGE acrylamide gel (Invitrogen). Following electrophoresis, gels were immunoblotted with anti-PGAM5 antibody. c Comparison of myc-tagged ∆23 PGAM5 filaments in transiently transfected COS7 cells stained with anti-myc antibody using three different imaging methods: confocal microscopy (left panel), stimulated emission depletion (STED) microscopy (middle panel), and structured illumination microscopy (SIM) (right panel). All scale bars correspond to 5 µm. d SIM images of myc-tagged ∆23 PGAM5 filaments in COS7 cells transiently expressing wild type (WT) or the R288E PGAM5 (red). Nuclei were stained using DAPI (blue). Both scale bars in d correspond to 10 µm
