Fig. 3

NIPSNAP1 promotes c-Myc protein stability as part of a mutual regulatory feedback loop. A Western blotting analysis of c-Myc levels in HCT116 and HepG2 cells after transduction with sh-Ctrl or two independent-shRNAs targeting sh-NIPSNAP1. Actin was used as a loading control throughout. B Parallel assays conducted in the cells from A measuring c-Myc mRNA levels using qPCR. C Cycloheximide (CHX) chase assays comparing the stability of c-Myc in HCT116 cells after transduction with sh-Ctrl or sh-NIPSNAP1 lentiviruses. The cells were pretreated with 50 mg/ml CHX for 0–4 h before analysis of NIPSNAP1 and c-Myc expression levels by Western blotting. As indicated, the effects of co-treating cells with or without 10 mM MG132 were determined at the 4 h timepoint. D Analysis of the polybiquitylation of ectopically expressed c-Myc. HCT116 (left) or HepG2 (right) cells were transduced with sh-Ctrl or sh-NIPSNAP1 before co-transfection with hemagglutinin (HA)-Ub and 3*Flag-c-Myc. Cell lysates (input) were subjected to Western blotting against NIPSNAP1 and a GAPDH loading control whereas anti-Flag immunoprecipitates (IPs) were blotted for anti-Ub or anti-HA as shown. E Ubiquitination analysis of 3*Flag-c-Myc measured as per D in HCT116 cells co-transfected without or with sh-NIPSNAP1 in combination with either HA-Ub WT, HA-Ub K48R, or HA-Ub K63R, respectively. F HCT116 cells transduced with sh-Ctrl or sh-NIPSNAP1 in combination with two different concentrations of 3*Flag-c-Myc were subjected to Western blotting to measure c-Myc, NIPSNAP1 and P27 (top) or SA-β-gal staining (bottom), respectively. B is mean ± SD; n = 3 independent experiments, two-tailed Student’s t test, Images in A and C–F represents three independent experiments (*P < 0.05; **P < 0.01, ***p < 0.001)