Figure 4. RNF8 controls septin SEPT7 ubiquitylation. (A) 293 cells were co-transfected with GFP-RNF8 or GFP-RNF8C403S and Myc-SEPT7, as indicated. Cells were lysed 48 h later and immunoprecipitated with anti-Myc (anti-Myc IP). Samples were analyzed by blotting with anti-Ub or anti-GFP. (B) 293 cells were siRNA treated and 48h later were transfected with a plasmid encoding Myc-SEPT7 and grown for a further 48 h. Immunoprecipitates were prepared as in (A) then blotted with indicated antibodies (C). As (A) but instead of Myc-SEPT7, cells were co-transfected with empty plasmid or FLAG-tagged SEPT2, 4, 8 or 11. (D) Quantification of cellular anomalies in RNF8 depleted cells or in cells treated with two independent siRNAs targeting SEPT7. All experiments were independently repeated multiple times; unpaired t-test (**, p < 0.01; ***, p < 0.001).