Fig. 4: CAMK2D associates with RNF8’s FHA domain weakly/transiently via p-Thr287 to mediate RNF8-induced mitotic checkpoint.
From: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma
A Volcano plot showing the proteins which are significantly depleted in *FHA vs RNF8 proximal proteomes (i.e., minus log2fold change (FC)). B Western blot analysis of endogenous CAMK2D levels in the streptavidin pull down lysates of HEK293T overexpressing BirA*-RNF8 or *FHA. CAMK2D/myc ratio was normalized to BirA*-RNF8 group. Western blot analysis of flag-CAMK2D levels in the streptavidin pull down lysates (C), or flag IP (D) of HEK293T co-transfected with flag-CAMK2D and BirA*-RNF8/*FHA. The band of interest is indicated by *. E Western blot analysis of flag levels in streptavidin pulldown lysates from HEK293T cell lysates overexpressing flag-CAMK2D or -CAMK2DT287A mutant, along with BirA*-RNF8. F Phos-tag SDS-PAGE analysis of flag-CAMK2D and -CAMK2DT287A mutant, along with co-expression of EV or myc-tagged RNF8/*FHA. The p-T287/flag ratio was normalized to empty vector control. G Western blot analysis of H3 pS10 levels in GFP or RNF8 overexpressing GSC TS543, with or without CAMK2D KD. RNF8 and CAMK2D serve as positive control whereas GAPDH serve as the loading control. H Western blot analysis of H3 pS10 levels in wildtype CAMK2D or CAMK2DED overexpressing GSC TS543, with or without RNF8 KD. Flag and RNF8 serve as the positive controls, while GAPDH serves as the loading control.
