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. 2019 Apr 15;159-160:146–156. doi: 10.1016/j.ymeth.2019.02.005

Fig. 3.

Fig. 3

Analysis of RNAPII degradation. (A) Comparison of different RPB1 antibodies for detection of UV-induced RPB1 degradation. Identical samples (lysates from HEK293 TRex flpIn cells, either untreated or irradiated with 20 J/m2 UVC and collected 3 h post-irradiation), were run in quadruplicate on the same gel, and the membrane was cut and probed with different antibodies, recognising either total RPB1 (D8L4Y), all forms of RPB1 (with preference for the phosphorylated form) (4H8), Ser 5-phosphorylated RPB1 (3E8), or Ser 2-phosphorylated RPB1 (3E10). (B) Dose-dependent RPB1 degradation upon UV irradiation. HEK293 TRex flpIn cells were irradiated with 5, 10 or 20 J/m2 UVC, and samples were taken at different timepoints after irradiation. (C) Use of the proteasome inhibitor MG132 to confirm proteasomal degradation of RPB1. HEK293 TRex flpIn cells were pre-treated with 5 μM MG132 for 3 h, UV irradiated with 20 J/m2 UVC, and samples were collected 3 h after irradiation.