Ab5095

Manufactured by Merck Group

Ab5095 is a laboratory equipment product offered by Merck Group. It is designed for general laboratory use. The core function of Ab5095 is to provide a reliable and consistent platform for various experimental procedures. No further details on the intended use or specific applications of this product are available.

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Lab products found in correlation

Ab4729, by Abcam (2 mentions) Ab11826, by Thermo Fisher Scientific (1 mentions) A11008, by Thermo Fisher Scientific (1 mentions) Ab63365, by Abcam (1 mentions) A10037, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Ab137078, by Abcam (1 mentions) Ab74073, by Abcam (1 mentions) Sc 13119, by Santa Cruz Biotechnology (1 mentions) Pan acetyl h3, by Merck Group (1 mentions) Ab5131, by Merck Group (1 mentions) Ni sepharose 6 fast flow, by GE Healthcare (1 mentions) Glass bottom dish, by NEST Biotechnology (1 mentions) Ab5408, by Abcam (1 mentions) Anti γ tubulin, by Merck Group (1 mentions) Airyscan mode, by Zeiss (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)

5 protocols using ab5095

Immunofluorescence Microscopy of SC35 and Associated Nuclear Factors

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Coverslips of T cells were permeabilized by incubation with 1% Triton X-100 for 20 min and probed with a mouse antibody to phospho-epitopes of human SC35p (Abcam, Cambridge, UK; ab11826) followed by a secondary goat antibody to mouse Alexa-Fluor 568 (Lifetech A-10037). To examine colocalization, T cells were probed with an antibody mix of primary mouse antibody to SC35 with rabbit-raised antibodies targeting either human RNA-Pol-II-ser2p (Abcam ab5095), H3K4me3 (Merck 07-473), H3K27ac (Abcam ab4729), or PKC-θ (Abcam AB63365) followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488 (Lifetech A-11008), respectively.
Coverslips were subsequently mounted on glass microscope slides with ProLong Gold anti-fade reagent (Life Technologies). Confocal laser scanning microscopy was used to study SC35 localization as previously described (35 (link)). 0.5 μm-spaced images were obtained with a Nikon x60 oil immersion lens on a Nikon C1 plus confocal system using NIS-Elements AR 3.2 software. The final image was obtained by averaging four sequential images from the same section at high resolution.

McCuaig R.D., Dunn J., Li J., Masch A., Knaute T., Schutkowski M., Zerweck J, & Rao S. (2015). PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation. Frontiers in Immunology, 6, 562.

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Comprehensive Antibody Panel for Cell Signaling

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Rabbit anti-CREB serum (244, in-house), rabbit anti-CRTC1 (C71D11, 2587, CST), rabbit anti-CRTC2 serum (6865, in-house), rabbit anti-CRTC3 (C35G4, 2720, CST), rabbit anti-CRTC3-pSer³⁹¹ (PBL #7408, in-house; Sonntag et al., 2019 (link)), rabbit-anti-hCRTC3 (PBL #7019, in-house; Sonntag et al., 2019 (link)), rabbit anti-mOCA2 (PBL #7431, in-house, this work), rabbit anti-H3AcK27 (ab4729, Abcam), Anti-RNA polymerase II (CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)), mouse anti-Tubulin (05-829; EMD Millipore), mouse anti-MITF (clone C5, MAB3747-1 Millipore; MITF ChIP grade ab12039 Abcam), rabbit anti-TYR (ab61284, Abcam), rabbit anti-DCT (ab74073, Abcam), rabbit anti-MLANA (NBP254568H, Novus), rabbit anti-PMEL (ab137078, Abcam) rabbit anti-ERK1/2 (4695, CST), rabbit anti-pERK1/2 (9101, CST), mouse anti-HSP90α/β (SC-13119, Santa Cruz Biotechnology), rabbit anti-Histone H3 (9715, CST).

Ostojić J., Yoon Y.S., Sonntag T., Nguyen B., Vaughan J.M., Shokhirev M, & Montminy M. (2021). Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by integrating cAMP and MAPK/ERK pathways. Cell reports, 35(7), 109136.

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ChIP Assays and qPCR Analysis

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ChIP assays and analysis of fractions by qPCR were performed as previously validated and described in detail [77 (link)]. Primer sequences are shown in Table 2. Frozen tissue samples were finely ground in liquid nitrogen prior to the fixation and lysis procedure. Antibodies against the following epitopes were used: normal rabbit IgG control (Millipore, Burlington, MA, #12–370), panacetyl-H3 (Millipore #06599MI), trimethyl-H3Lys4 (Millipore #0473MI), RNA polymerase II CTD repeat (phospho-Ser5, Abcam, Cambridge, MA, #ab5131), RNA polymerase II CTD repeat (phospho-Ser2, Abcam #ab5095) and PRDM16 (Sigma Aldrich #SAB3500989). Bound:input ratios were determined by the comparative C_t value method as Bound/Input=2^{(input Ct-bound Ct)} using a 1% v/v aliquot of input chromatin.

Patel B.V., Yao F., Howenstine A., Takenaka R., Hyatt J.A., Sears K.E, & Shewchuk B.M. (2020). Emergent Coordination of the CHKB and CPT1B Genes in Eutherian Mammals: Implications for the Origin of Brown Adipose Tissue. Journal of molecular biology, 432(23), 6127-6145.

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Antibodies Generation and Validation

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Pol II S2P and Pol II S5P antibodies were purchased from Sigma-Aldrich (Ab5095 and Ab5131). For the ChIP-Seq analysis of CBP/Neijre, Rpb3, Spt5, NELF-A and NELF-E we used previously described antibodies^{2 ,3 (link)}. Antibodies against NELF-B (epitope corresponding to 150–594 amino acid residues) were generated in this study. The epitopes for antibody production were expressed as 6 × His-tagged fusion proteins in Escherichia coli, affinity-purified on Ni Sepharose 6 Fast Flow (GE Healthcare), according to the manufacturer’s protocol, and injected into rabbits, following the standard immunization procedure. Antibodies were affinity-purified using the same epitopes as were used for immunization. The specificity of antibodies against EcR, Spt5, NELF A and NELF B was characterized in the RNA interference experiments by the specific depletion of corresponding proteins (Figs. S1–S4). Antibodies production was performed according to procedures outlined in the NIH (USA) Guide for the Care and Use of Laboratory Animals. The protocol used was approved by the Committee on Bioethics of the Institute of Gene Biology of the Russian Academy of Sciences. All procedures were performed under conditions designed to minimize suffering.

Mazina M.Y., Kovalenko E.V, & Vorobyeva N.E. (2021). The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes. Scientific Reports, 11, 172.

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Immunofluorescence and Live-Cell Imaging

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Cells were quickly rinsed with pre-warmed DPBS, xed with pre-cooling methanol for 10 min at -20°C, and permeabilized in 0.5% Triton X-100 for 10 min. Cells were then blocked with 5% Bovine Serum Albumin for 30 min and incubated overnight at 4°C with anti-RNAPII phospho S5 (1:1500, Abcam, ab5408), anti-RNAPII phospho S2 (1:3000, Abcam, ab5095), anti-γ-Tubulin (1:1000, Sigma, T6557 For live imaging, cells in glass bottom dish (NEST, 801001) were transfected with pEGFP-Tubulin and mCherry-H2B using Lipofectamine 3000 (Invitrogen), and synchronized with double thymidine block procedure. Cells were then released into thymidine-free medium for 8 hours and imaged in PeCon environmental microscope incubator (ZEISS) at 37°C and 5% CO 2 . Images were collected on the LSM 880 confocal microscope using a 63× oil immersion objective lens with Airyscan mode (ZEISS).

Yuan K., Liu H., Yu C., Zhang N., Yang M., Can-Hua H., Hu C., Chen F., Hu X., & Zhang Z. (2021). Elevated pausing of RNA Polymerase II underlies acquired resistance to ionizing radiation.

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