Figure 4.

SIAH1 promoted ubiquitination and proteasomal degradation of CPSF1

(A and B) The interaction between SIAH1 and CPSF1 in 22Rv1 cells was determined by reciprocal co-IP assay. (C and D) The protein level of CPSF1 in 22Rv1 cells was assessed by CHX pulse-chase assay in the presence or absence of SIAH1 overexpression. CHX (50 μg/mL) was used for inhibiting the de novo protein synthesis and then relative protein density of CPSF1 was assessed at different time points (0, 2, 3, and 6 h). (E and F) CHX (50 μg/mL) was used for inhibiting the de novo protein synthesis and then relative protein density of CPSF1 was assessed at different time points (0, 3, and 6 h) in LNCaP cells. (G) The ubiquitination of CPSF1 was assessed by CPSF1 IP followed by western blot with ubiquitin antibody. (H) Western blot analysis for CPSF1 protein in 22Rv1 cells after SIAH1 overexpression in the presence or absence of MG132 (20 μM). (I) The interaction of SIAH1-CPSF1 was verified by PLA assay in 22Rv1 cells.