Fig. 3

Lysine (K) 74 crotonylation of SEPT2 was identified. A Scatter diagram based on the fold change (FC) of protein expression (FC < 2) and crotonylation (FC > 1.5). B Validation of endogenous crotonylation of SEPT2, GRB2 and RAB35. MHCC-97L cells were treated with 25 mM sodium crotonate (NaCr) for 24 h. Cell lysates were immunoprecipitated with corresponding primary antibodies, followed by western blotting (WB) with an anti-pan-Kcr antibody. C Validation of endogenous differences in SEPT2, GRB2 and RAB35 crotonylation in MHCC-97H and MHCC-97L cell lines. SEPT2 crotonylation was highest crotonylation in the MHCC-97H cells. D The location of differentially crotonylated sites in the SEPT2 domain. E SEPT2-K74 is evolutionarily conserved in seven species. K74 in SEPT2 is highlighted in red. F The SEPT2-K74 and SEPT2-K318 mutants were crotonylated to a lesser extent than wild-type (WT) SEPT2. The degree of crotonylation in cells with ectopically expressed Flag-tagged SEPT2 WT, K74R and K318R was analyzed. G GTPase activity was impaired after SEPT2-K74R mutation. Flag-tagged SEPT2 WT and K74R proteins were purified, and an in vitro GTP hydrolysis assay was performed. Panels 1, 2, 4, and 5 show the negative controls, and panel 3 shows the positive control. H Western blot analysis of overexpressed Flag-tagged SEPT2 WT and K74R, pan-crotonylation and SEPT2 K74-crotonylation (K74-cr) in cells and crotonylation on SEPT2 with or without NaCr treatment