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

Figure EV1. p97 regulates RNF8 turnover.

Figure EV1

  1. Western blot analysis showing increased RNF8 protein level in HeLa cells by CHX chase after siRNA‐mediated p97 depletion.
  2. Quantification of (A) showing reduced RNF8 degradation rate after siRNA‐mediated p97 depletion.
  3. Western blot analysis of CHX chase in HEK293 cells showing reduced RNF8 degradation rate after doxycycline‐inducible mild expression of p97EQ (dominant negative ATPase inactive) variant.
  4. Western blot analysis of Flag‐RNF8 denaturing‐IP showing RNF8 K48‐Ub, K63‐Ub and total‐Ub (HA) ubiquitination pattern in soluble pool (cytosol + nucleosol) and on chromatin under physiological conditions and after IR (10 Gy).
  5. Western blot analysis showing comparison of Ub‐K48 and Ub‐K63 antibodies against different amounts (0.25, 0.5 and 1.0 μg) of purified recombinant K48‐ or K63‐Ub chains (Ub‐2 to Ub‐7).
  6. Western blot analysis showing distribution of different proteins under physiological conditions and after IR (10 Gy) in cytosol, nucleosol, loosely bound chromatin (LB Chro) and tightly bound chromatin (TB Chro) of HEK293 cells.
  7. Representative IF images showing presence of ATX3 in the nucleus of U2OS cells (scale bar: 10 μm). Nuclei are marked by white lines.

Source data are available online for this figure.