Pbrm1

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PBRM1 is a protein-coding gene that is responsible for producing the PBRM1 protein, which is a subunit of the SWI/SNF chromatin remodeling complex. The PBRM1 protein plays a role in regulating gene expression by modifying the structure of chromatin.

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Anti-PBRM1 (6 citations) Anti-PBRM1 (A301–591A (2 citations)

4 protocols using pbrm1

Antibody Characterization for Chromatin Remodeling

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The following antibodies were utilized: SMARCA2 (Cell Signaling, 11966, dilution 1:2000), SMARCA4 (Abcam, ab110641, dilution 1:1000), PBRM1 (Bethyl Labs, A301-591A, dilution 1:1000), SMARCC1 (Cell Signaling, 11956, dilution 1:1000), HDAC1 (Cell Signaling, 34589, dilution 1:1000), VHL (Cell Signaling, 68547, dilution 1:1000), FLAG (Sigma, F3165, dilution 1:1000), Lamin A/C (Cell Signaling, 4777, dilution 1:1000), α−Tubulin (Sigma, T6074, dilution 1:1000), β-Tubulin (Cell Signaling, 2128, dilution 1:1000), β-Actin (Cell Signaling, 3700, 1:1000 and 4970, dilution 1:1000) and total ubiquitin (Cell Signaling, 3936, dilution 1:1000).

Cantley J., Ye X., Rousseau E., Januario T., Hamman B.D., Rose C.M., Cheung T.K., Hinkle T., Soto L., Quinn C., Harbin A., Bortolon E., Chen X., Haskell R., Lin E., Yu S.F., Del Rosario G., Chan E., Dunlap D., Koeppen H., Martin S., Merchant M., Grimmer M., Broccatelli F., Wang J., Pizzano J., Dragovich P.S., Berlin M, & Yauch R.L. (2022). Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers. Nature Communications, 13, 6814.

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Western Blot Analysis of SWI/SNF Proteins

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Total protein was denatured for 10 min at 95 °C, separated on a 4–12% SDS-polyacrylamide gel, and transferred to a PVDF membrane (Immobilon FL, EMD Millipore, Billerica, MA). The membrane was blocked with 5% bovine serum albumin (VWR, Batavia, IL) in PBS containing 0.1% Tween-20 (PBST) for 30 mins at room temperature and then incubated in primary antibodies overnight at 4°C. The primary antibodies used were directed against ARID1A (Santa Cruz Biotechnology Inc., Dallas, TX; sc-32761), PBRM1 (Bethyl Laboratories, Montgomery, TX; A301–591A), BAF155 (Santa Cruz Biotechnology Inc., Dallas, TX; Sc-32763), BAF47 (Santa Cruz Biotechnology Inc., Dallas, TX; Sc-166165), LAMIN B1 (Santa Cruz Biotechnology Inc., Dallas, TX; Sc-377000). The primary antibodies were detected by incubating the membranes in goat-anti-rabbit or goat-anti-mouse secondary antibodies (LI-COR Biotechnology, Lincoln, NE) conjugated to IRDye 800CW or IRDye 680 respectively for 1 h at room temperature, and the signals were visualized using Odyssey Clx imager (LI-COR Biotechnology, Lincoln, NE).

Marian C.A., Stoszko M., Wang L., Leighty M.W., de Crignis E., Maschinot C.A., Gatchalian J., Carter B.C., Chowdhury B., Hargreaves D.C., Duvall J.R., Crabtree G.R., Mahmoudi T, & Dykhuizen E.C. (2018). Small Molecule Targeting of Specific BAF (mSWI/SNF) complexes for HIV Latency Reversal. Cell chemical biology, 25(12), 1443-1455.e14.

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Cell Culture and Reagent Protocol for NSCLC Cell Lines

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PC9 cells were kindly provided by J. Engelman (Massachusetts General Hospital Cancer Center and Harvard Medical School). H1975 cells were kindly provided by Kwok Kin Wong (Dana-Farber Cancer Institute and Harvard Medical School). 293T cells were purchased from American Type Culture Collection (ATCC). PC9 cells/H1975 cells and 293T cells were maintained in RPMI 1640 medium (ATCC modification) and DMEM, respectively (Thermo Fisher Scientific, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GE Healthcare HyClone), 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Gefitinib, erlotinib, and AZD9291 were purchased from Selleck Chemicals. Palbociclib was a gift from K. Cichowski. The antibodies used were Tubulin (1:2500; Cell Signaling, 2128S), GAPDH (1:2500; Cell Signaling, 8884), PBRM1 (1:500; Bethyl Laboratories, A301-591A-M), p21 (1:1000; Cell Signaling, 2947S), phospho-EGFR (Tyr1068; 1:1000; Cell Signaling, 2236S), EGFR (1:1000; Cell Signaling, 2232S), phospho-ERK1/2 (T202/Y204; 1:1000; Cell Signaling, 4370P), total ERK1/2 (1:1000; Cell Signaling, 4695P), phospho-AKT (S473; 1:1000; Cell Signaling, 4060P), total AKT (1:1000; Cell Signaling, 2920S), ETV1 (1:1000; Abcam, ab184120), ETV5 (1:1000; Abcam, ab54704), and CIC (1:5000; a gift from H. Zoghbi).

Liao S., Davoli T., Leng Y., Li M.Z., Xu Q, & Elledge S.J. (2017). A genetic interaction analysis identifies cancer drivers that modify EGFR dependency. Genes & Development, 31(2), 184-196.

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Knockout Cell Lines Viability Assay

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We purchased HAP1 knockout cell lines from Horizon Discovery in August 2015 (Cambridge, UK) and the knockouts were confirmed using Western blot analysis. Cells were plated in triplicate at a density of 2500 cells/well, and grown in complete IMDM glutamax media (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin, in 5% CO₂ at 37°C. We treated the cell lines with varying doses of docetaxel and paclitaxel. Cell viability was evaluated after 3, 4, and 8 days of treatment using CellTitre-Glo® (Promega) luminescent assay on a Victor X5 plate reader (Perkin Elmer). We used commercial antibodies: SMARCA4 (Santa Cruz sc-17796), SMARCA2 (Abcam ab15597), ARID1B (Abcam ab57461, gamma-tubulin (Sigma T5326), ARID1A (Santa Cruz sc-32761), ARID2 (Santa Cruz sc-166117), PBRM1 (Bethyl A-301-591A-M), and Lamin B1 (Abcam ab16048). We used Memcode Protein Stain Kit (Thermo Scientific) to detect transferred proteins to nitrocellulose membranes. We used secondary antibodies conjugated with Horseradish peroxidase (Interchim) and revealed the signal by chemiluminescence substrate from Pierce (SuperSignal West Pico).

Gurard-Levin Z.A., Wilson L.O., Pancaldi V., Postel-Vinay S., Sousa F.G., Reyes C., Marangoni E., Gentien D., Valencia A., Pommier Y., Cottu P, & Almouzni G. (2016). Chromatin regulators as a guide for cancer treatment choice. Molecular cancer therapeutics, 15(7), 1768-1777.

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