Fig. 5: CUL3–KCTD10 directly ubiquitinates TCEA2 to remodel the RNAP complex.
From: KCTD10 is a sensor for co-directional transcription–replication conflicts
a, Denaturing Ni-NTA pulldown from HEK293T cells expressing Flag-tagged TCEA2 and His-tagged ubiquitin (His–Ub) and treated with 1 μM ICRF193 or DMSO in the presence of 50 μM MG132 for 4 h. b, Denaturing Ni-NTA pulldown from KCTD10-deficient (sh3) and control HEK293T cells expressing wild-type or K184R mutant Flag–TCEA2 and/or His–Ub in the presence of 50 μM MG132. c, Western blot of the chromatin fraction from KCTD10-deficient or control U2OS cells treated with 1 μM ICRF193, 1 μM etoposide (ETP) or 2.5 μM VCP inhibitor (VCPi; NMS-873) for 6 h. d, Western blot of chromatin fractions from TCEA2-deficient HEK293T cells expressing wild-type TCEA2, TCEA2(K184R) or empty vector (−) treated with ICRF193 (1 μM) or DMSO for 4 h. siTCEA2, small interfering RNA (siRNA) targeting TCEA2. e, EdU incorporation in KCTD10-deficient (sh3), TCEA2-deficient (siTCEA2) and control U2OS cells treated with 10 μM EdU and 100 nM ICRF193 or control for 6 h. Data are mean ± s.d. (n = 3 biologically independent experiments). siCtr, control siRNA. f,g, γH2AX foci from KCTD10-deficient U2OS cells with control siRNA (siCtr) or siTCEA2 and treated with 100 nM ICRF193 or DMSO for 6 h. f, Quantification of foci. Data are mean ± s.d. (n = 200 cells examined over 3 independent experiments). g, Representative images. Scale bars, 10 μm. Data are mean ± s.d. P values were calculated with one-way ANOVA with Bonferroni’s multiple comparisons test.
