Fig. 5

Dissecting interactions between NIPSNAP1, FBXL14 and c-Myc. A Western blotting analysis of the levels of NIPSNAP1, FBXL14, Ki67, c-Myc, and P27 in HepG2 cells subjected to transduction with sh-Ctrl (−) or sh-NIPSNAP1 alone or in combination with sh-FBXL14. GAPDH was used a loading control throughout. B Ubiquitylation assays performed in HCT116 cells transduced with sh-Ctrl, sh-FBXL14 or sh-NIPSNAP1 in combination with co-transfection with hemagglutinin (HA)-Ub and 3*Flag-c-Myc. Cell lysates (input) were subjected to Western blotting against NIPSNAP1 and FBXL14 whereas anti-Flag immunoprecipitates (IPs) were blotted with anti-Flag and anti-HA, respectively. C HCT116 cells were transduced with 3*Flag-c-Myc in combination with sh-Ctrl or sh-NIPSNAP1 before Western blotting analysis of cell lysates or anti-Flag immunoprecipitates against Flag, FBXL14 and NIPSNAP1, respectively. D Schematic illustrating the design of full-length Flag-tagged FBXL14 (FBXL14-WT) and overlapping truncated constructs (FBXL14-P1, -P2, -P3, P4 and P5, respectively; left). HCT116 cells were transfected with the indicated constructs and subjected to immunoprecipitation with anti-Flag antibodies before Western blotting against Flag and NIPSNAP1, respectively (right). E, F HepG2 cells were subjected to transduction with sh-Ctrl (−) or sh-NIPSNAP1 alone or in combination with sh-FBXL14 before assessment of cell proliferation using CCK-8 assays (E) and cellular senescence as SA-β-gal staining (F). E Is mean ± SD; n = 3 independent experiments, two-tailed Student’s t test, images in A–D and F represents three independent experiments (*P < 0.05; **P < 0.01, ***p < 0.001)