anti rnap rpb1 mouse monoclonal antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: eIF4E and Ezrin cooperate in pseudopods to drive a localized migratory translation program in acute myeloid leukemia

doi: 10.64898/2026.02.21.707190

Figure Lengend Snippet: A. Western blot of total cell lysates from MM6 cells grown in suspension demonstrated knockdown of Ezrin (siEZR) or eIF4E (siEIF4E) compared to RNAi to luciferase (siLUC) used as a negative control. b-Actin is provided as a loading control. Other proteins of the Ezrin-CD44-HA axis are also shown. Quantification for these is shown in with 3-6 biological replicates for each protein. B. Adhesion and invasion capacity of MM6 cells onto/through HS-5 bone marrow stroma. Fold change relative to siLUC is shown. Each symbol represents a biological replicate performed independently with replicates. Bars represent the mean, shown with standard deviations and p-values (one-way ANOVA). C. Western blot of eIF4E and Ezrin immunoprecipitations (IPs) using total cell lysates from MM6 cells in suspension. SN, supernatant after immunoprecipitation, IgG, negative control. Representative of three biological replicates. IPs of total cell lysates from THP-1 cells in suspension are provided in . D. IPs from MM6 cells in suspension using the rRNA antibody Y10b. LC indicates antibody light chain. Representative of three biological replicates. E. RNA immunoprecipitations (RIPs) from MM6 total cell lysates grown in suspension using anti-Ezrin (Ezrin RIP) or anti-eIF4E (eIF4E RIP) antibodies. Data are from RT-qPCR and represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). F. Western blots of eIF4E and Ezrin IPs from the cytoplasmic fractions of MM6 cells in suspension or G . After invasion through HS-5 bone marrow stroma (invaded). Fractionation controls are provided in . H. RIPs from MM6 cytoplasmic fraction from invaded cells using anti-eIF4E (eIF4E RIP) or anti-Ezrin (Ezrin RIP) antibodies. Data are from RT-qPCR represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). I. Count of the number of pseudopods observed in suspension and invaded MM6 cells represented as a fraction relative to the total cells counted. Each symbol represents a biological replicate. Bars represent the mean, shown with standard deviations and p-values (two-way ANOVA). J . Immunofluorescence and confocal microscopy demonstrating eIF4E, Ezrin, CD44 and rRNA are localized to the same pseudopods (white arrows). All confocal micrographs represent a single section through the plane of the cell. Scale bar = 10 µm

Article Snippet: In case of Y10b IPs, 100 ml of Y10b mouse hybridoma supernatant (DSHB), or 10 mg od mouse IgG (cat# 12-371, Millipore) were bound to the beads 50 μL protein G conjugated superparamagnetic beads in 1 ml of NT2 buffer ON at 4°C, washed three times with NT2 buffer, added to 1 mg of total cell lysates, incubated overnight at 4°C with rotation and washed as described for IPs with rabbit antibodies.

Techniques: Western Blot, Suspension, Knockdown, Luciferase, Negative Control, Control, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Fractionation, Immunofluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

doi: 10.1101/2025.09.30.678750

Figure Lengend Snippet: A) Schematic of active P-TEFb release from 7SK-snRNP to promote RNA polymerase II (RNAPII) pause release in response to stress and growth stimuli. B) Protein levels of HEXIM1, LARP7 and Tubulin (loading control) in U2OS cells 48 h after siRNA transfection. nonT = non-targeting control siRNA. C) Approach for siRNA depletion or small molecule inhibitor treatment prior to DNA fibre labelling in presence of camptothecin (CPT, 10 μM) or hydroxyurea (HU, 200 μM). D) Replication fork speeds (IdU label) after CPT or HU treatment in presence of HEXIM1 (+) or nonT (-) siRNA. Data from 2 repeats. E) Replication fork speeds (IdU label) after CPT or HU treatment in presence of LARP7 (+) or nonT (-) siRNA. Data from 2-3 repeats. F) DRB inhibits P-TEFb and p38 MAPK inhibitor (P38i) inhibits 7SK-snRNP/P-TEFb dissociation. G) Replication fork speeds (IdU label) after CPT or HU treatment +/- p38i. Data from 3 repeats. H) Replication fork speeds (IdU label) after CPT or HU treatment +/- 100 μM DRB. Data from 2 repeats. I) Nascent chromatin capture proteomics in HeLa S3 cells shows limited enrichment or depletion of HEXIM1, LARP7 and MEPCE at ongoing (nascent/mature chromatin), stalled (3 mM HU, 30 min/untreated) or collapsed (1 μM CPT, 40 min + roscovinite/roscovitine) replication forks compared to PCNA or RAD51. Roscovitine was included to prevent new origin firing. Scatter graphs show aggregates and medians (black points) of independent repeats with overall median (line). Kruskal-Wallis with multiple comparisons test, **** p ≤ 0.0001; ns: not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C in mouse anti-DNA-RNA hybrid antibody (S9.6 hybridoma growth medium, ATCC HB-8730, 1:1,000) or mouse anti-dsDNA antibody (Abcam ab27156, 1:100,000) in sterile 5 % BSA/TBST, before being washed in TBST and incubated in goat anti-mouse HRP (Cell Signaling 7074, 1:5,000) 5 % milk/TBST for 1 hour at room temperature.

Techniques: Control, Transfection

Journal: bioRxiv

Article Title: The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

doi: 10.1101/2025.09.30.678750

Figure Lengend Snippet: A) Protein levels of CDK9 in soluble extract (SE) and nuclear pellet (NP) fractions after 2 h treatment with 10 μM CPT or 200 μM HU +/- HEXIM1 siRNA (48 h). B) Protein levels of CDK9 in SE and nuclear NP fractions +/- LARP7 siRNA as in A. C) Relative levels of SE (7SK-snRNP bound) versus NP (chromatin-bound) P-TEFb after treatment +/- HEXIM1 siRNA as in A. n=3. D) Relative levels of SE versus NP P-TEFb after treatment +/- LARP7 siRNA as in B. n=3. E) Representative images of nascent RNA labelling with 5-ethynyluridine (EU). DNA was detected using DAPI. Bars: 50 μm. F) Nuclear EU intensities after treatment with 10 μM CPT and EU for 20 or 50 min. n=1. G) Nuclear EU intensities after treatment with 10 μM CPT or 200 μM HU (2 h) +/- HEXIM1 siRNA. n=3. H) Slot blots of genomic DNA stained with S9.6 antibody (RNA:DNA hybrids) and double-stranded DNA (dsDNA; loading control) after treatment with 10 μM CPT or 200 μM HU (20 min) +/- HEXIM1 siRNA. RNase H treatment was used as control. I) RNA:DNA hybrid quantification as in E. n=4. J) Replication fork speeds (IdU label) after CPT or HU treatment overexpression of turboGFP-RNase H1 (+) or eGFP vector control (-). Data from 2 repeats. K) Approach for siRNA depletion and 10 μM CPT or 200 μM HU treatment prior to immunostaining. L) Percentages of cells with >10 γH2AX foci after 2 h CPT or HU +/- HEXIM1 siRNA. n=2-3. M) Percentages of cells with >9 53BP1 foci after 2 h CPT or HU +/- HEXIM1 siRNA. n=3. Scatter blots show aggregates and medians (black points) of independent repeats with overall median (line). ANOVA or Kruskal-Wallis with multiple comparisons test, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C in mouse anti-DNA-RNA hybrid antibody (S9.6 hybridoma growth medium, ATCC HB-8730, 1:1,000) or mouse anti-dsDNA antibody (Abcam ab27156, 1:100,000) in sterile 5 % BSA/TBST, before being washed in TBST and incubated in goat anti-mouse HRP (Cell Signaling 7074, 1:5,000) 5 % milk/TBST for 1 hour at room temperature.

Techniques: Staining, Control, Over Expression, Plasmid Preparation, Immunostaining

Journal: Journal of Virology

Article Title: Oropouche virus NSs protein suppresses host transcription by targeting the RNA polymerase II RPB1 protein

doi: 10.1128/jvi.01176-25

Figure Lengend Snippet: Altered phosphorylation status and stability of the RNA polymerase II RPB1 subunit upon expression of Oropouche virus NSs protein. ( A–C ) Western blot analysis of total cell lysates from Vero cells infected with wild-type (wt) OROV or rMP12 NSs variants. Cells were mock-infected or infected at an MOI of 5 and harvested at 8 hpi ( A ), 12 hpi ( B ), or 24 hpi ( C ). ( D ) Cells were similarly infected and harvested at 16 hpi. The blot shows the hyperphosphorylated (IIo) and hypophosphorylated (IIa) forms of the RNA polymerase II RPB1 subunit, as well as CTD phosphorylation at serine 2 (Ser 2) and serine 5 (Ser 5), are shown. Expression of OROV NSs, as well as Flag- or SF-tagged NSs proteins, was detected using an anti-OROV NSs peptide antibody.

Article Snippet: The following antibodies were used in this study: anti-Flag tag (DYKDDDDK) rabbit monoclonal antibody (Cell Signaling Technology, #14793), anti-Flag M2 mouse monoclonal antibody (Millipore Sigma, F3165), anti-Histone H3 rabbit polyclonal antibody (Cell Signaling Technology, #9715), anti-β-actin mouse monoclonal antibody (Proteintech, #66009-1-Ig), anti-GAPDH mouse monoclonal antibody (Invitrogen, MA5-15738), anti-OROV mouse immune ascitic fluid (ATCC: VR-1228AF), anti-RVFV mouse immune ascitic fluid (WRCEVA, UTMB), anti-phosphorylated RNAP II CTD (Ser5) mouse monoclonal antibody (Thermo Fisher Scientific, #91119), anti-phosphorylated RNAP II CTD (Ser2) rabbit monoclonal antibody (Thermo Fisher Scientific, MA5-33187), anti-RNAP RPB1 mouse monoclonal antibody (Novus Biologicals, NB200-598), and anti-B23 (nucleophosmin 1 [NPM1]) mouse monoclonal antibody (Millipore Sigma, B0556).

Techniques: Phospho-proteomics, Expressing, Virus, Western Blot, Infection

Journal: Journal of Virology

Article Title: Oropouche virus NSs protein suppresses host transcription by targeting the RNA polymerase II RPB1 protein

doi: 10.1128/jvi.01176-25

Figure Lengend Snippet: Stabilizing effect of the proteasomal inhibitor MG-132 on the RNA polymerase II RPB1 subunit during Oropouche virus NSs protein expression. Vero cells were either mock-infected or infected with wild-type (wt) OROV, rMP12-ORONSs, or rMP12-ORONSs-C-Flag at an MOI of 5. A 10 µM concentration of MG-132 was added to the culture at 4 hpi. Cells were harvested at 16 hpi, and the levels of the RNAP II RPB1 subunit, as well as its phosphorylated forms at serine 2 (Ser 2) or serine 5 (Ser 5) within the CTD, were analyzed. OROV NSs protein was detected using an anti-OROV NSs peptide antibody.

Article Snippet: The following antibodies were used in this study: anti-Flag tag (DYKDDDDK) rabbit monoclonal antibody (Cell Signaling Technology, #14793), anti-Flag M2 mouse monoclonal antibody (Millipore Sigma, F3165), anti-Histone H3 rabbit polyclonal antibody (Cell Signaling Technology, #9715), anti-β-actin mouse monoclonal antibody (Proteintech, #66009-1-Ig), anti-GAPDH mouse monoclonal antibody (Invitrogen, MA5-15738), anti-OROV mouse immune ascitic fluid (ATCC: VR-1228AF), anti-RVFV mouse immune ascitic fluid (WRCEVA, UTMB), anti-phosphorylated RNAP II CTD (Ser5) mouse monoclonal antibody (Thermo Fisher Scientific, #91119), anti-phosphorylated RNAP II CTD (Ser2) rabbit monoclonal antibody (Thermo Fisher Scientific, MA5-33187), anti-RNAP RPB1 mouse monoclonal antibody (Novus Biologicals, NB200-598), and anti-B23 (nucleophosmin 1 [NPM1]) mouse monoclonal antibody (Millipore Sigma, B0556).

Techniques: Virus, Expressing, Infection, Concentration Assay