FIGURE 4:

FX12 directly interacts with RNF5 and inhibits its E3 activity in vitro. (A–C) SPR analysis. Purified cytosolic tail of RNF5 (RNF5N) was immobilized on a sensor chip. The binding sensorgrams were obtained when FX12 (A), FX41 (B), or FX36 (C) at the indicated concentrations were flowed across the sensor chip. (D) Differential effects on NHK-drGFP by FX36 and FX41. (E) FX12 inhibits autoubiquitination of GST fusion of RNF5N but not GST-Hrd1C and -praja1 In vitro. (F) FX12 does not affect ubiquitin charging to E1 (UBA1) in vitro. (G) FX12 does not affect ubiquitin charging to E2 (UbcH5B) in vitro. (H, I) Transient overexpression of the RNF5 RING finger mutant (RINGm) inhibits NHK degradation (H). The graph (right) shows relative band intensities of NHK measured by ImageJ and normalized to actin. The band intensities of NHK at time 0 were set as 100%. Error bars = SEM for n = 3 experiments. The p value was calculated from a one-tailed Student’s t test. *p < 0.01.