Fig. 3.

Lysine deficient mutant pVHL were resistant to degradation therefore increased half-life. a Schematic diagrams for various lysine deficient VHL mutants used in this study. b HA tagged lysine deficient mutant VHL were transfected into HEK293T and/or Flag-UCP. At 48 h post-transfection, cells were harvested and analyzed by western blotting. c Each lysine mutated pVHL expressing plasmids were transfected in HEK293T. At 36 h post- transfection. Cells were treated with cyclohexamide (CHX) for 0, 2, 4, 6 h then and immunoblotted as indicated. d 786-O cell lines constitutively expressing lysine mutant (K159, K171, K196, Lysine zero) stable cell lines were treated with cyclohexamide (CHX) for 0, 2, 4, 6 h then and immunoblotted as indicated. Calculation of protein degradation kinetics of HEK293T (E) and 786-O (f) cell lines showed that half-life of lysine deficient pVHL were increased (n = 3, p < 0.01)