Fig. 4

NIPSNAP1 sequesters FBXL14 to inhibit c-Myc degradation. A Fast Silver Staining gel comparing anti-Flag immunoprecipitants from HCT116 cells transduced with either a control vector (pCDH) or Flag-tagged NIPSNAP1 construct (pCDH-NIPSNAP1-Flag). The highlighted bands denoted as FBXL14 and NIPSNAP1, respectively, were assigned based on mass spectrometry (MS/MS) analysis tabulated elsewhere as Additional file 8: Table S6. B Western blotting-based assessment of interactions between endogenous NIPSNAP1 and candidate ubiquitin modifiers detected in A and from the literature associated with c-Myc regulation. Only FBXL14 was recovered within NIPSNAP1 immunoprecipitates (IPs) from HCT116 cells. GAPDH was used as a loading control throughout. C Endogenous NIPSNAP1 is recovered within FBXL14 IPs from HCT116 cells conducted as per B. D Representative confocal images showing immunofluorescence staining against NIPSNAP1 (green), FBXL14 (red), and DAPI (blue) in HepG2 cells (bar = 20 µm). E Western blotting analysis of FBXL14 levels in HepG2 cells after transduction with sh-Ctrl or two independent-shRNAs targeting NIPSNAP1. F Western blotting analysis of the levels of Ki67, NIPSNAP1, c-Myc and P27 in HCT116 cells after transduction with sh-Ctrl or two independent-shRNAs targeting FBXL14. G Western blot analysis of HCT116 cells as per F after transduction with graded concentrations of FBXL14-MYC. Ectopic FBXL14 levels were detected with anti-Myc epitope antibodies. H Cycloheximide (CHX) chase assays comparing the stability of c-Myc in HCT116 cells after transduction with sh-Ctrl or sh-FBXL14 lentiviruses. The cells were pretreated with 50 mg/ml CHX for 0–24 h before analysis of FBXL14 and c-Myc expression levels by Western blotting. I Analysis of the effects of FBXL14 on the polybiquitylation of c-Myc. HCT116 cells were transduced with FBXL14-MYC and co-transfected with hemagglutinin (HA)-Ub and 3*Flag-c-Myc. Input samples or anti-Flag IPs were then subjected to Western blotting against HA, Flag and MYC, respectively. J Ubiquitination of 3*Flag-c-Myc measured as per I in cells co-transfected with FBXL14-MYC in combination with either HA-Ub WT, HA-Ub K48R, or HA-Ub K63R, respectively. K The stability of the indicated Flag-tagged lysine substitution mutants of c-Myc in the absence (empty vector) and presence of ectopically expressed FBXL14-MYC was measured in HCT116 cells. The expression of c-Myc and FBXL14 was revealed by Western blotting using antibodies against Flag and MYC, respectively. L Ubiquitylation assays performed in HCT116 cells after individually transfecting WT Flag-tagged c-Myc, or the indicated substitution mutants together with HA-Ub and FBXL14-MYC. Cell lysates (input) were subjected to Western blotting against MYC and a GAPDH loading control whereas anti-Flag immunoprecipitates (IPs) were blotted with anti-Flag and anti-HA, respectively