rpb1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: The nuclear oncoprotein SET is necessary for MLL/KMT2A binding and transcriptional elongation

doi: 10.64898/2026.02.26.708410

Figure Lengend Snippet: A: IGV snapshot depicting ChIP profiles of Mll-N, H3K4me3, RNAPolII (total), RNAPolII-Ser5P, and RNAPolII-Ser2P at the promoter of the Set target Med23 . B: Metagene plots across all Set activated genes reveal a specific loss of Mll and elongating RNA polymerase from chromatin after Set depletion. C: Only promoter recruited PP2A can repress the MLL responsive human Meis1 promoter. The reporter construct was co-transfected with expression constructs as indicated and luciferase results were determined in triplicate experiments. D: Recruitment of Set and PP2A to the endogenous Meis1 promoter in myeloid precursor cells. Effectors were targeted to the murine Meis1 promoter in MLL-ENL transformed cells by a fusion with catalytically inactive Cas9 (dCas9) in combination either with a non-targeting (vec) or two Meis1 promoter specific sgRNAs. Meis1 output was measured by RT-qPCR in triplicates.

Article Snippet: Set : Invitrogen (#MA5-35772); MLL: CST (#8178) mixed 1:1 with anti-MLLN, Upstate (Temecula, CA, #05-764) 10 μl AB-mix per 5×10e6 cells, H3K4me3: CST (#9751), RNAPolymeraseII: CST (#14958), RNAPolII-Ser5P: CST (#13499), RNAPolII-Ser2P: CST (#13523), Msk1-P (Ser376P): CST (#9591), H3S10P: CST (#53348), H3K9ac: CST (#9649), H3K14ac: CST (#7627), H3K27ac: CST (#8173), Cbp: CST (#7389), Creb-P (Ser133P): CST (#9198), MOZ: Invitrogen (PA5-68046) mixed 1:1 with Invitrogen (PA5-103467), HA: CST (#3724), flag: Merck (F1804).

Techniques: Construct, Transfection, Expressing, Luciferase, Transformation Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Hierarchical mechanisms control the clearance of DNA lesion–stalled RNA polymerase II

doi: 10.1038/s41467-026-68413-4

Figure Lengend Snippet: a Schematic of local DRB run-off assay by immunofluorescence imaging. Cells were treated with 100 μM DRB, preventing new RNAPII molecules from going into elongation, immediately after local UV-C (100 J/m2), then fixed after 0, 1, or 2 h and stained by immunofluorescence. b Representative images of ( a ), stained for RNAPII-S2P (Abcam, Ab5095) in RPE1 WT cells at the indicated time points after local UV-C, marked by CPD (Cosmo Bio, CAC-NM-DND-001). Scale bar, 10 μm. c Quantification of ( b ). RNAPII-S2P mean pixel intensity inside local damage was divided by that outside local damage in the same cells to correct for the general RNAPII run-off caused by DRB. The experiment was performed three times ( n = 3). Each transparent colored circle represents one cell, each outlined solid circle the mean of two technical replicates ( > 50 cells / technical replicate). Black lines represent the means of three independent experiments. d Schematic of global DRB run-off assay by western blotting. RNAPII was detected by western blot from whole cell lysates of RPE1 WT cells at 0, 1, and 2 h after global UV-C (30 J/m 2 ) and incubation with 100 μM DRB. e Representative image of ( d ). The antibody against RNAPII-S2P (Millipore, 04-1571 (3E10)) detects only elongating RNAPII (upper blot). The antibody against the N-terminal domain of RNAPII (Cell Signaling, 14958 (D8L4Y)) detects both initiating (RNAPIIa) and elongating (RNAPIIo) forms (middle blot). HSPA4 was used as loading control. f Quantification of blots in ( e) RNAPII-S2P mean pixel intensity was divided by that of HSPA4 and normalized to 0 h. g RNAPII-S2P + UV / -UV ratio of quantification in ( f ). The value of each UV-treated time point in ( f ) was normalized to its respective unirradiated condition. h Representative images of local DRB run-off with and without proteasome inhibitor (MG132; 20 µM; 1 h pretreatment and the indicated incubation times after UV-C). i Quantification of ( h ) as described in ( c ). j Representative image of global DRB run-off assay with and without proteasome inhibitor as described for ( h ). HDAC1 was used as loading control. RNAPII antibodies are as in ( e ) . k Quantification of ( j ) as described in ( f ) and ( g ).

Article Snippet: The antibody against the N-terminal domain of RNAPII (Cell Signaling, 14958 (D8L4Y)) detects both initiating (RNAPIIa) and elongating (RNAPIIo) forms (middle blot).

Techniques: Immunofluorescence, Imaging, Staining, Western Blot, Incubation, Control

Journal: Nature Communications

Article Title: Hierarchical mechanisms control the clearance of DNA lesion–stalled RNA polymerase II

doi: 10.1038/s41467-026-68413-4

Figure Lengend Snippet: a Top: Cartoon depicting the effect of UVSSA KO on TCR complex assembly. Bottom: Quantification of local DRB run-off assay as described for Fig. RPE1 WT and UVSSA KO with or without VCPi treatment (NMS-873; 5 µM; 1 h pretreatment, then incubation for the indicated times after UV-C). b Representative images of RNAPII-S2P signal detected by western blot from whole cell lysates of primary fibroblasts from normal (48BR) or UVSSA-defective cells (Kps3) at 0, 2, 4, and 6 h after global UV-C (10 J/m 2 ). Cells were incubated with VCPi (CB5083; 10 µM) for 1 h before UV-C and incubated in the same media. RNAPII-S2P was detected with an antibody against phosphorylated C-terminal domain (CTD) Ser2/5 (BioLegend, 920203 (H5)). Short and long exposures are shown (top and middle panels, respectively). SMC3 was used as loading control. This experiment was repeated three times. c Detection of RNAPII-S2P ubiquitylation (Cell Signaling, 3936) and TFIIH recruitment (p89 and p62) by western blot after immunoprecipitation of RNAPII-S2P (right) from chromatin fractions (left) of RPE1 WT and indicated TCR KOs. Cells were incubated with or without VCPi (NMS-873; 5 µM) for 1 h before 12 J/m 2 UV-C. Then, cells were incubated in the same media and collected 1 h after UV-C. This experiment was repeated three times. d Detection of RNAPII-S2P ubiquitylation (Cell Signaling, 3936) and TFIIH recruitment (p89 and p62) by western blot after immunoprecipitation of RNAPII-S2P (right) from the chromatin fraction (left) of RPE1 WT cells or UVSSA KO with or without complementation of the indicated UVSSA mutants. Cells were treated with 12 J/m 2 UV-C and collected after 1 h. This experiment was repeated three times. e Top: Cartoon depicting the effect of UVSSA ΔTFIIH on TCR complex assembly. Bottom: Quantification of the local DRB run-off immunofluorescence assay as described for Fig. in RPE1 WT, UVSSA KO , and UVSSA KO stably expressing the indicated UVSSA constructs and with or without VCPi treatment as in ( a ). f Top: Cartoon depicting the effect of STK19 KO on TCR complex assembly. Bottom: Quantification of the local DRB run-off immunofluorescence assay as described for Fig. in RPE1 WT and STK19 KO with or without VCPi treatment as in ( a ).

Article Snippet: The antibody against the N-terminal domain of RNAPII (Cell Signaling, 14958 (D8L4Y)) detects both initiating (RNAPIIa) and elongating (RNAPIIo) forms (middle blot).

Techniques: Incubation, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Stable Transfection, Expressing, Construct