Figure 1. RNF8 is a ubiquitinated substrate of p97 under physiological and genotoxic conditions.

1. Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA‐mediated p97 depletion under physiological conditions and after IR (10 Gy).
2. Graph represents the quantifications of (A) (***P < 0.001; unpaired t‐test, n = 3, mean + SEM).
3. Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10 μM for 6 h) under physiological conditions and after IR (10 Gy).
4. Graph represents the quantifications of (C) (**P < 0.01, ***P < 0.001; unpaired t‐test, n = 3, mean + SEM).
5. Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline‐inducible mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).
6. Graph represents the quantifications of (E) (**P < 0.01, ****P < 0.0001; unpaired t‐test, n = 4, mean + SEM).
7. Model representing the processing of ubiquitinated substrate by p97 ATPase activity. Inactivation of p97 ATPase activity leads to the accumulation of ubiquitinated substrate.
8. Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA‐mediated p97 depletion.
9. Graph represents the quantifications of (H) (***P < 0.001, ****P < 0.0001; two‐way ANOVA, n = 3, mean + SEM) and Western blot for efficacy of siRNA depletion of p97 (right).
10. Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing K48‐linked hyper‐ubiquitination of RNF8 after siRNA‐mediated p97 depletion.
11. Western blot analysis of Strep‐p97 Co‐IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97‐WT under physiological conditions and after IR (10 Gy).
12. Graph represents the quantifications of (K) (*P < 0.05; unpaired t‐test, n = 2, mean + SEM).

Source data are available online for this figure.