Figure 4.

Binding of HEXIM1 to the P-TEFb component Cyclin-T1 is reduced, and the C-terminal domain of RNAP II is hyperphosphorylated in patient cells. (a) Endogenous Cyclin-T1 was immunoprecipitated from lysates obtained from patient-, control 1- and patient’s mother-derived fibroblasts. For control purposes, lysates from all three samples were pooled and incubated with an IgG isotype control antibody (“Pool”). Immunoprecipitates (IP) and total cell lysates (Input) were analysed by immunoblotting using the indicated antibodies. The band representing the antibody heavy chain is indicated with a star. Representative blots are shown. Full-length blots are presented in Fig. S5. (b) Band intensities were quantified with a chemiluminescence imager. The amount of co-immunoprecipitated HEXIM1 was normalized to the precipitated amount of Cyclin-T1. The mean of five independent experiments ± SD is given. **p ≤ 0.01, ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. (c) Immunoblot of lysates obtained from patient, control 1 and patient’s mother fibroblast cultures from three different passages. The amount of total and phosphorylated RNAP II (RNAP II-S2P) was monitored with specific antibodies, and anti-Tubulin antibody was used to control for equal loading. Representative blots are shown. Full-length blots are presented in Fig. S6. (d) Quantification of phosphorylated RNAP II. Band intensities were quantified with a chemiluminescence imager. The amount of RNAP II-S2P was normalized to total RNAP II. The mean of five independent experiments ± SD is given. ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison.