Fig. 4

USP14 stabilizes PFKL by promoting its deubiquitination. A USP14 was depleted by shRNAs in the OSCC cells and the indicated proteins were examined by Western blotting. B USP14 was ectopically overexpressed in the OSCC cells and the indicated proteins were examined by Western blotting. C HN4 and HN6 cells were cultured in the absence or presence of b-AP15 (1 μM) for 24 h. Cellular extracts were collected for Western blotting with indicated antibodies. D, E qRT-PCR analysis of the indicated genes in HN4 cells with USP14 knockdown or overexpression. F Increasing amounts of HA-USP14 plasmids were co-transfected with Flag-PFKL plasmid into HEK293T cells and the indicated proteins were examined by Western blotting. G HEK293T cells were transfected with HA-USP14 or vector plasmids. A CHX chase experiment was performed and Flag-tagged PFKL protein was determined by Western blotting (left panel). The right panel showcases the relative protein amounts of different groups. Error bars represent ± S.D. of triplicate experiments. H HEK293T cells were transfected with HA-Ub and Flag-PFKL plasmids, immunoprecipitation (IP) was performed using Flag antibody. The indicated proteins were examined by Western blotting. I HEK293T cells were transfected with HA-Ub, Flag-PFKL and HA-USP14 plasmids, IP was performed using Flag antibody. The indicated proteins were examined by Western blotting. J HN4 and HN6 cells were treated with b-AP15 (1 μM) for 24 h and further incubated with or without MG132 (5 μM) for another 5 h. The indicated proteins were examined by Western blotting