Fig. 3: CDK9 and CTDP1 are associated with RPB7 in RPB1 stabilization regulation.

a Schematic of transcription inhibitor treatment in RPB7 degron cells is shown. b Western blotting analyses for RPB7 degron cells, treated with the indicated range of dosages of the THAL-SNS-032 (dCDK9), flavopiridol (Flav) or DRB for 2 h and then with 500 μM auxin for 3 h. β-Actin served as a loading control. The bottom bar graph showing the relative protein level of hypophosphorylated RPB1 as means (n = 2, biological replicates). c Heatmap showing the enrichment of Pol II related phosphatases in ChIP-MS of the twelve subunits of Pol II. d The schematic diagram on the left shows TurboID tag at the C-terminus of RPB1 will label the proximally interacting proteins upon adding the biotin, then the biotin-labeled proteins were enriched by streptavidin beads to detected by label-free MS. The flow diagram on the right shows the TurboID-MS assay we did in RPB7 degron cells. e Up: Volcano plot showing the protein enrichment changes identified by TurboID-MS upon RPB7 degradation. The red dot indicates CTDP1, blue dots indicate other subunits of Pol II, and yellow dots indicate subsets of Pol II related phosphatases. The heatmap shows the log₂FC of Pol II subunits. Bottom: A network showing the phosphatases enriched in RPB7 ChIP-MS. The color and size of the circles both indicate the log2(IP/GFP) enrichment score. The border color of the circles indicates the log2FC of these phosphatases in RPB1 TurboID-MS upon RPB7 degradation. The p value was analyzed with two-sided t-test with standard deviation moderated by empirical Bayes method in Limma software. f Co-immunoprecipitation (Co-IP) assay. Lysate of CTDP1 or RPB7 degron cells were immunoprecipitated with antibodies against either GFP (“GFP” lane) or rabbit IgG (“IgG” lane). In total, 5% of cell lysate without antibodies was loaded as a control (“Input” lane). Left: Western blot analyses of anti-GFP immunoprecipitates collected from lysate of CTDP1-GFP degron cells. Right: Reverse co-IP of RPB7 in RPB7-GFP degron cells is shown. g Co-IP of FLAG in knock-in RPB7-Halo-Flag cells after auxin treatment. Levels of enriched RPB1, RPB4, and CTDP1 were examined.