Smarca4

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Sourced in United States, Ireland, China

SMARCA4 is a protein that is a subunit of the SWI/SNF chromatin remodeling complex. It is responsible for the ATP-dependent remodeling of chromatin structure, which is a crucial process for the regulation of gene expression.

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SMARCA4/BRG1 (3 citations) Anti-SMARCA4 (2 citations)

12 protocols using smarca4

Nuclear Protein Interactome Analysis

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Immunoprecipitations (IP) were performed with 100 ug of nuclear extract resuspended in standard IP buffer (150 mM NaCl, 1 mM EDTA, 1% Triton-X) and rotated overnight with 1.25 ug antibody. Antibodies used were: SMARCA4 (Santa Cruz, G-7; Abcam ab110641 used in Supplementary Figure 2F); SMARCA2, (Bethyl A301–015); SMARCC1 (Santa Cruz, H-76); SMARCD1 (Santa Cruz, 23); ARID2 (Santa Cruz, E-3); PBRM1 (EMD/Millipore, ABE70, Bethyl, A301–591A); ARID1A (Santa Cruz, C1), SS18 (Cell Signaling Technologies, D6I4Z), SMARCC2 (Santa Cruz, E-6), and mouse IgG (Santa Cruz, sc-2025) as a negative control.

Pan J., McKenzie Z.M., D’Avino A.R., Mashtalir N., Lareau C.A., St. Pierre R., Wang L., Shilatifard A, & Kadoch C. (2019). The ATPase module of mammalian SWI/SNF family complexes mediates subcomplex identity and catalytic activity-independent genomic targeting. Nature genetics, 51(4), 618-626.

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Western Blot Analysis of Chromatin Proteins

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Loading buffer (Lane Marker Reducing Sample Buffer
Thermo-Scientific) was added to 5 μg or 1 μg of nuclear or
chromatin protein respectively and the sample denatured at 95° C for
5 min, spun briefly and kept on ice. The samples and a molecular weight
marker (Spectra Multicolor Broad Range Protein Ladder, Invitrogen) were
loaded in a 4–20 % polyacrylamide Amersham ECL Gel (GE Healthcare,
UK). The run was performed at a constant 160 mV in Tris – Glycine
Running Buffer. The electrophoresed proteins were transferred to a
nitrocellulose membrane (Pierce) in a Tris-Glycine-Ethanol Transfer Buffer,
by application of an electrical field of 100 mV for 45 min. Then they were
incubated in blocking solution.
The membrane was incubated in a solution composed of primary
antibodies in T-TBS (BAF47 1:1000, 612110 BD Biosciences; SMARCA4 1:1000,
sc-10760 SantaCruz, H3K27me3 1:20000, C15410196 Diagenode; H3K27ac 1:20000,
C15410196 Diagenode; H3 1:20000, ab1791 Abcam; B- actin:20000, ab49900
Abcam; Anti-Mouse IgG 1:10000, ab6728 Abcam; Anti-Rabbit IgG 1:10000,
ab205718 Abcam; ) for 1 hour at room temperature. It was washed three times
in a solution of T-TBS then incubated in a diluted specific secondary
antibody (1:10000) for 1 hour at room temperature and washed in T-TBS as
before. The blot was developed using SuperSignal West Pico Chemiluminescent
Substrate (Pierce) and imaged using ChemiDoc XRS+ (Biorad).

Erkek S., Johann P.D., Finetti M.A., Drosos Y., Chou H.C., Zapatka M., Sturm D., Jones D.T., Korshunov A., Rhyzova M., Wolf S., Mallm J.P., Beck K., Witt O., Kulozik A.E., Frühwald M.C., Northcott P.A., Korbel J.O., Lichter P., Eils R., Gajjar A., Roberts C.W., Williamson D., Hasselblatt M., Chavez L., Pfister S.M, & Kool M. (2018). Comprehensive analysis of chromatin states in atypical teratoid/rhabdoid tumor identifies diverging roles for SWI/SNF and Polycomb in gene regulation. Cancer cell, 35(1), 95-110.e8.

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SWI/SNF Chromatin Remodeling Complex Analysis

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Protein was loaded onto Bis-Tris 4–12% gradient Novex gels and run for 150V for 90 minutes. A wet transfer was performed for 2.5 hours at 165 mA at 4 degrees Celsius on to PVDF membrane. After transfer, membranes were blocked in milk for 1 hour at room temperature before applying primary and fluorescent secondary antibodies (Goat Anti-Mouse IgG Antibody, IRDye® 680RD Conjugated, LICOR, Goat Anti-Rabbit IgG Antibody, IRDye® 800CW Conjugated, LICOR) for visualization on a LICOR Odyssey. Antibodies used were: SMARCA4 (Santa Cruz, G-7); SMARCA2, (Bethyl A301–015); SMARCC1 (Santa Cruz, H-76); SMARCC2 (Santa Cruz, E-6); SMARCD1 (Santa Cruz, 23); ARID2 (Santa Cruz, E-3); PBRM1 (EMD/Millipore, ABE70); ARID1A (Santa Cruz, C7), SS18 (Cell Signaling Technologies, D6I4Z), BRD7 (Santa Cruz, B-8), TBP (Abcam, 5184), SMARCB1 (Santa Cruz, A-5), GAPDH (Santa Cruz, G-9), ACTL6A (Santa Cruz, E-3), and CTCF (EMD/Millipore, 07–729).

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Protein Extraction and Immunoblotting of PDX Tumors

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Tumor samples from frozen tissue were available for cases 101, 102, and 113 (patient-derived xenograft tumor). Before protein extraction, hematoxylin and eosin-stained frozen section slides were prepared from this tissue and examined by a gynecologic pathologist to ensure tumor purity. The proteins were extracted using RIPA Lysis buffer supplemented with protease and phosphatase inhibitors. After 15 min of incubation on ice, the lysates were spun for 15 min at 20 000 g in the cold Microfuge. Protein concentrations were measured by Bradford. Extracted proteins were resolved on SDS-PAGE, transferred onto nitrocellulose, and blotted for antibodies targeting proteins of interest: SMARCA4 (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-10768, dilution 1:1000), SMARCA2 (Cell Signaling Technology, Danvers, MA, USA; #11966, dilution 1:1000), and β-Actin (Santa Cruz Biotechnology, sc-69879, dilution 1:10 000).

Jelinic P., Schlappe B.A., Conlon N., Tseng J., Olvera N., Dao F., Mueller J.J., Hussein Y., Soslow R.A, & Levine D.A. (2015). Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type. Modern Pathology, 29(1), 60-66.

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Immunoblotting of SWI/SNF Complex

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After indicated treatments, cell lysates were harvested using RIPA buffer (Cell Signaling Technologies) with protease inhibitors (cOmplete, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Antibodies used were as follows: ARID1A (Santa Cruz; sc-373784), α-tubulin (Santa Cruz; sc-5286), β-Actin (C-4) (Santa Cruz; sc-47778), β-Actin (Cell Signaling; 8457), BAF57/SMARCE1 (Bethyl Laboratories, A300-810A), BAF60a (Santa Cruz; sc-135843), BAF155 (Cell Signaling; 11956), BAF170 (Santa Cruz; sc-166237), SMARCA4 (Santa Cruz; 17798), Cleaved Caspase-3 (Cell Signaling; 9664), c-MYC (Santa Cruz; sc-764) or c-MYC (Cell Signaling; 9402), cyclin B1 (Cell Signaling; 4135 and 4138), cyclin D1 (Santa Cruz; sc-718), GAPDH (Cell Signaling; 2118S and 97166S), GRP78 (Rockland Antibodies, Limerick, PA; 200–301 F36), IRE1-alpha (Cell Signaling; 3294), lamin A/C (Cell Signaling; 2032), PSMB5 (Abcam, Cambridge, MA; ab3330), SMARCB1/SNF5 (Bethyl A301-087A), UBE2C (Proteintech, Rosemont, IL; 66087–1). Results shown are representative of at least two biological replicates.

Hong A.L., Tseng Y.Y., Wala J.A., Kim W.J., Kynnap B.D., Doshi M.B., Kugener G., Sandoval G.J., Howard T.P., Li J., Yang X., Tillgren M., Ghandi M., Sayeed A., Deasy R., Ward A., McSteen B., Labella K.M., Keskula P., Tracy A., Connor C., Clinton C.M., Church A.J., Crompton B.D., Janeway K.A., Van Hare B., Sandak D., Gjoerup O., Bandopadhayay P., Clemons P.A., Schreiber S.L., Root D.E., Gokhale P.C., Chi S.N., Mullen E.A., Roberts C.W., Kadoch C., Beroukhim R., Ligon K.L., Boehm J.S, & Hahn W.C. (2019). Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition. eLife, 8, e44161.

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Targeted Silencing of Chromatin Remodelers

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RNAi knockdown of SMARCA4, SMARCA2, ARID1A and ARID1B was done by Lipofectamine 2000 (Invitrogen) transfection of Dharmacon ON-TARGETplus siRNA pools (20nM final), in comparison to Non-Targeting Control (NTC) siRNA pool. Previous studies demonstrated on-target knockdown, as two or more individual siRNAs from each pool exhibited similar knockdown efficiency and cellular phenotype [7 (link)]. Re-expression of SMARCA4 was done by retroviral transduction (pBABE-puro-SMARCA4) [7 (link)], in comparison to empty vector control. Knockdown and/or re-expression were confirmed by western blotting of whole cell lysates, using the following primary antibodies: SMARCA4 (sc-17796; Santa Cruz), SMARCA2 (sc-166579; Santa Cruz), ARID1A (ab176395; Abcam), ARID1B (ab57461; Abcam), and GAPDH (sc-25778; Santa Cruz). Knockdown efficiency was quantified using ImageJ.

Davidson J., Shen Z., Gong X, & Pollack J.R. (2017). SWI/SNF aberrations sensitize pancreatic cancer cells to DNA crosslinking agents. Oncotarget, 9(11), 9608-9617.

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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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SMARCA2/SMARCA4 Double Knockdown Protocol

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SMARCA4 and SMARCA2 double knockdown was carried out by siRNA transfection directed against SMARCA2 (siRNA-SMARCA2):
5 ′ -UCGUCGAGCAAUCAUUUGGUU-3 ′ and (siRNA-SMARCA4) SMARCA4:
5 ′ -UAGCAUUGAGGGCUGUCUCCA-3 ′ . A nontargeting control siRNA (siRNA-NC): 5 ′ -AACCACGUGAGGCAUCCAGGC-3 ′ was used as a negative control. For double knockdown, individual siR-NAs of SMARCA2 and SMARCA4 were co-transfected into cells with DR-GFP insertion 24 hours before HA-I-SceI-GR pCW tet-on plasmid transfection. The siRNA transfections were carried out using Lipofectamine RNAiMAX (Invitrogen). The full-length siRNA-resistant SMARCA2 was cloned into S protein-FLAG-Streptavidin binding peptide (SFB) vector. The K755A mutant of SMARCA2 was generated by site-directed mutagenesis from the siRNA-resistant full-length SMARCA2. Re-expression of full-length SMARCA2 or the ATPase inactive mutant (K755A) was performed by plasmid transfection using Lipofectamine 2000 (Thermo Fisher Scientific) 20 hours before HA-I-SceI-GR pCW tet-on plasmid transfection. Knockdown and/or re-expression were confirmed by Western blotting using the following primary antibodies: SMARCA4 (Catalog number: sc-17796; Santa Cruz, Shanghai, China), SMARCA2 (Catalog number: sc-166579; Santa Cruz, Shanghai, China), Vinculin (Catalog number: A14193; Abclonal, Wuhan, Hubei, China).

Yu L, & Wu D. (2024). SMARCA2 and SMARCA4 Participate in DNA Damage Repair. Frontiers in bioscience (Landmark edition), 29(7).

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Tumor Protein Extraction and Western Blot

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Tumor samples from frozen tissue were available for cases 101, 102, and 113 (patient-derived xenograft tumor). Prior to protein extraction, hematoxylin and eosin (H&E)-stained frozen section slides were prepared from this tissue and examined by a gynecologic pathologist to ensure tumor purity. The proteins were extracted using RIPA Lysis buffer supplemented with protease and phosphatase inhibitors. After 15 minutes of incubation on ice, the lysates were spun for 15 minutes at 20,000×g in the cold Microfuge. Protein concentrations were measured by Bradford. Extracted proteins were resolved on SDS-PAGE, transferred onto nitrocellulose, and blotted for antibodies targeting proteins of interest: SMARCA4 (Santa Cruz Biotechnology, Dallas, TX; #sc-10768, dilution 1:1000), SMARCA2 (Cell Signaling Technology, Danvers, MA; #11966, dilution 1:1000), and β-Actin (Santa Cruz, sc-69879, dilution 1:10 000).

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Western Blot Analysis of Key Neurogenic Proteins

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Western blots were subsequently blocked in 5% BLOTTO (Santa Cruz Biotechnology #NC9730946) dissolved in Tris-buffered saline with 0.1% Tween 20 detergent (TBS-T), incubated in primary antibody overnight at 4°C, washed 3 times in TBS-T, and probed with secondary antibody for 1–2 h at RT. The following antibodies were used in this study: SMARCA4 (Santa Cruz Biotechnology #sc-374197, RRID:AB_10990135), SMARCA2 (Abcam #ab15597, RRID:AB_443214), beta-actin (GeneTex #GTX629630, RRID:AB_2728646), CCND1 (Abcam #ab134175, RRID:AB_2750906), PHOX2B (Santa Cruz Biotechnology #sc-376997, RRID:AB_2813765), ISL1 (DSHB #40.2D6, RRID:AB_528315), HAND2 (Abcam #ab200040, RRID:AB_2923502), GATA3 (Thermo Fisher #MA1-028, RRID:AB_2536713), MYCN (Active Motif #61185, RRID:AB_2793543), ASCL1 (Santa Cruz Biotechnology #sc-374104, RRID:AB_10918561), EBF3 (Thermo Fisher #PA5-30985, RRID:AB_2548459), HRP-conjugated mouse anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology #sc-2357, RRID:AB_628497) and m-IgGk BP-HRP secondary antibody (Santa Cruz Biotechnology #sc-516102, RRID:AB_2687626). Detection of HRP-conjugated antibodies was performed using Clarity (Bio-Rad #1705060) or Clarity Max Western ECL substrate (Bio-Rad #1705062) on a Bio-Rad ChemiDoc MP imager.

Cermakova K., Tao L., Dejmek M., Sala M., Montierth M.D., Chan Y.S., Patel I., Chambers C., Loeza Cabrera M., Hoffman D., Parchem R.J., Wang W., Nencka R., Barbieri E, & Hodges H.C. (2023). Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF. Nucleic Acids Research, 52(1), 4-21.

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