Fig 1: Protein stability of ERa is enhanced in cells with AKTIP loss through CAND1(A) MCF7 cells stably expressing AKTIP shRNA or vector were treated with 10 µg/mL cycloheximide (CHX) for the indicated hours before being subjected to western blotting. ERK2 was loading control. The graph shows band intensities quantified by densitometry in ImageJ and normalized to that at 0 h (n = 3). *p < 0.05 using two-way ANOVA for comparison between groups with Sidak’s multiple comparison test.(B) Lysates of AKTIP-depleted or vector control cells were subjected to immunoprecipitation (IP) with anti-ERa antibody before western blotting (immunoblotting [IB]). ERa protein levels were normalized prior to IP by using proportionally different amounts of lysates. IP with normal immunoglobulin G (IgG) was control.(C) AKTIP-bound proteins pulled down in MCF7 cells were identified by mass spectrometry. Protein interaction network of the protein partners was generated by STRING. ERa, which was not pulled down in the IP, was included in the STRING analysis to reveal proteins that potentially interact with ERa. Only proteins in the ubiquitin/proteasome degradation pathway are shown in the network map. Red dots: hits known to inhibit protein ubiquitination. Line thickness reflects the degree of confidence of the interaction.(D–G) MCF7 cells stably expressing AKTIP shRNA or vector were transfected with (D) a pool of 4 siRNA of each gene indicated, (E and F) individual siRNA of CAND1, or (G) OX plasmid of CUL2 or NEDD8 for 72 h. Protein lysates were harvested for western blotting or IP.(H) Lysates of AKTIP-depleted or vector control cells were subjected to IP with anti-CUL2 antibody (top) or anti-CAND1 antibody (bottom) using equal amounts of lysates.(I) Lysates of AKTIP-depleted or vector control cells were subjected to IP with anti-ERa antibody using proportionally different amounts of lysates for equal ERa levels (top) or anti-CUL2 antibody using equal amounts of lysates (bottom). IP with IgG was control. Input was lysate without the IP procedure. Relative densitometric values (after normalization with the immunoprecipitated proteins) are provided below the blots. *p < 0.05; ***p < 0.001; ns, no significant difference compared with mock using t test (n = 3). The western blots shown are representatives of three independent experiments.