Fig. 5: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex.
From: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma
A Schematic diagram to illustrate the workflow for sucrose density gradient ultracentrifugation to assess RNF8 protein distribution in various fractions. B Western blot analysis of myc-RNF8, flag-CAMK2D, and MAD2 levels, with or without CAMK2D overexpression, in various fractions after sucrose density gradient ultracentrifugation of 293T cell lysates. C Western blot analysis of myc-RNF8 levels, with or without CAMK2D overexpression, in pooled fractions 6 to 8 from (B). The myc/input ratio was normalized to the EV control. D Western blot analysis of myc-RNF8/*FHA, flag-CAMK2D and MAD2 levels, with CAMK2D overexpression, in various fractions after sucrose density gradient ultracentrifugation of 293T cell lysates. E Western blot analysis of myc-RNF8/*FHA and MAD2 levels, with CAMK2D overexpression, in pooled fractions 6–8 from (D). The myc/input ratio was normalized to the RNF8 control. F Western blot analysis of flag-RNF8 and HA-MAD2 levels in the streptavidin pull down lysates of HEK293T overexpressing BirA*-CAMK2D, along with or without flag-RNF8 and HA-MAD2 overexpression. G Western blot analysis of flag-CAMK2D levels in the streptavidin pulldown lysates of BirA*-RNF8 or GFP-overexpressing HEK293T, with or without 200 ng/ml NOC (16 h).
