Skip to main content
. 2019 Oct 15;38(21):e102361. doi: 10.15252/embj.2019102361

Figure 2. Homeostasis of RNF8 is controlled by auto‐ubiquitination and the ubiquitin–proteasome system.

Figure 2

  1. Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing the ubiquitination pattern of RNF8‐WT and RNF8‐RING* variant, under siRNA‐mediated luciferase (siLuc) or p97‐depleted conditions (sip97).
  2. Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10 μM).
  3. Graph represents the quantifications of (B) (ns: not significant, P > 0.05, ****P < 0.0001; two‐way ANOVA, n = 2, mean + SEM).
  4. Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing hyper‐ubiquitination of RNF8 after proteasome inhibition (MG132, 10 μM for 6 h).
  5. Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag‐RNF8‐WT and Flag‐RNF8‐RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.
  6. Graph represents the quantifications of (E) (ns P > 0.05, ****P < 0.0001; two‐way ANOVA, n = 3, mean + SEM).

Source data are available online for this figure.