Fig. 2
HectH9 promotes K63-linked ubiquitination of HK2. a PC-3 cell lysates were harvested for immunoprecipitation (IP) with IgG or anti-HectH9 antibody, followed by immunoblot (IB) analysis with indicated antibodies. The ratio of immunoprecipitated HK2 to total HK2 levels in the input was quantified using ImageJ software. Arrowhead indicates the full-length of HectH9 and signals underneath represent HectH9 fragments. b PC-3 cell lysates were harvested for IP with IgG or anti-HK2 antibody, followed by IB analysis with indicated antibodies. c and d In vivo ubiquitination assay of HK2 by HectH9. HEK293T cells transfected with indicated plasmids were harvested for ubiquitination assay. Cells were lysed by denatured buffer. The ubiquitinated proteins were precipitated with nickel (Ni)-NTA beads and subjected to IB analysis. NTA indicates nickel bead precipitate; WCE indicates whole-cell extracts. e HEK293 cells with Luciferase or HectH9 knockdown were transfected with indicated constructs and harvested for in vivo ubiquitination assay. f Purified catalytically active form (WT) or defective mutant (C4341A) of GST-HectH9 proteins were incubated with adenosine triphosphate, E1 (Ube1) and E2 (Ubc13), with or without purified Flag-HK2 for in vitro HK2 ubiquitination assay. g HEK293T cells transfected with indicated constructs were harvested for in vivo ubiquitination assay. h IB analysis of HK2 and HK1 expression in PC-3 cells with GFP or HectH9 knockdown. Two HectH9 lentiviral shRNAs were used in this assay. The relative intensity of protein expression as indicated was quantified with ImageJ software and normalized to α-Tubulin expression. Immunoblots were performed three times
