Fig. 2: TTC36 Modulates c-Myc Expression and Ubiquitylation.
A Immunoblotting of liver lysates from wild-type (WT) and TTC36−/− C57BL/6 mice using antibodies against TTC36 and c-Myc; β-Actin served as the loading control. B Western blotting of TTC36 knockdown PLC/PRF/5 or TTC36 knockout HepG2 cells was performed using c-Myc antibody; β-actin was a loading control. C Immunoblotting of PLC/PRF/5 or Huh7 cells transfected with a plain vector or TTC36-encoding plasmid using indicated antibodies. D, E Q-PCR analysis of TTC36 and c-Myc mRNA levels in PLC/PRF/5, normalized to 18S, with error bars representing mean ± SD (n = 3). *p < 0.05, **p < 0.01. F, G Western blot analysis of PLC/PRF/5 cells with TTC36 knockdown or HepG2 with TTC36 knockout, treated with or without MG-132 (10 μM) for 6 h at indicated time points. H, I Western blot analysis of PLC/PRF/5 or Huh7 cells with Flag-TTC36 overexpression, treated with or without MG-132 (10 μM) for 6 h at indicated time points. J, K Immunoblotting analysis of PLC/PRF/5 cells expressing Flag-TTC36 or TTC36 knockdown, treated with 10 μg mL-1 CHX at indicated time points. L Western blot analysis of PLC/PRF/5 or Huh7 cells with or not with si-TTC36 and based on stable Flag-TTC36 overexpression, PLC/PRF/5 or HepG2 cells with or not with Flag-TTC36 overexpression and based on si-TTC36, to observe changes in c-Myc protein levels. M Western blot analysis of PLC/PRF/5 cells with c-Myc knockdown or Flag-TTC36 overexpression, or co-transfection, to observe changes in c-Myc protein levels. N HEK-293T cells transfected with GFP-TTC36 or si-TTC36 in addition to overexpressing His-Ub and Flag-c-Myc, treated with MG-132 (10 μM) for 6 h, and subjected to immunoprecipitation with Ni-NTA affinity resin followed by immunoblotting with indicated antibodies.
