Western blot analysis was performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in the RIPA buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the specific primary antibodies, followed by peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence (Denville Scientific). The following antibodies were used: antibodies to E-cadherin (1∶1000, BD Transduction Laboratories, 610182), vimentin (1∶2000, NeoMarkers, MS-129-P), Erbb2 (1∶500, Cell signaling Technology, 2242), HOXA1 (1∶1000, Santa Cruz Biotechnology, sc-17146), SMARCA5 (1∶500, sc-8760 from Santa Cruz Biotechnology and ab3749 from Abcam), SMARCD1 (1∶500, Abcam, ab86029), mTOR (1∶1000, Cell signaling Technology, 2972), BMPR2 (1∶1000, Cell signaling Technology, 69679), cyclin D1 (1∶1000, Cell signaling Technology, 2922), ALDH1A1 (1∶1000, Santa Cruz Biotechnology, sc-22589), HSP90 (1∶3000, BD Transduction Laboratories, 610419) and cyclophilin B (1∶2000, Thermo, PA1-027A).
Ab3749
Manufactured by Abcam
Sourced in United States
Ab3749 is a lab equipment product from Abcam. It is designed for general laboratory use. The core function of this product is to perform a specific task in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab63801, by Abcam (2 mentions)
Ab16048, by Abcam (2 mentions)
Precast gradient gel, by Bio-Rad (1 mentions)
Bmpr2, by Cell Signaling Technology (1 mentions)
E cadherin, by BD (1 mentions)
Erbb2, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ab1790, by Abcam (1 mentions)
4336 apc 050, by Cell Signaling Technology (1 mentions)
Universal magnetic co ip kit, by Active Motif (1 mentions)
Ab1012, by Abcam (1 mentions)
Ab10158, by Abcam (1 mentions)
Anti myc antibody sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti xpc antibody, by Santa Cruz Biotechnology (1 mentions)
Anti cpd antibody, by Cosmo Bio (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Anti acetyl histone h4 antibody, by Merck Group (1 mentions)
Ab36999, by Abcam (1 mentions)
7 protocols using ab3749
1
Comprehensive Western Blot Analysis
2
Fractionation and Immunoprecipitation of Nuclear Protein Complexes
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To resolve nuclear multiprotein complexes, nuclear extracts from S1 cells (23 (link)) were loaded onto a 10–40% sucrose gradient and ultracentrifuged for 40 h at 4°C and 214 000 g. Fractions of equal volumes were precipitated with trichloroacetic acid and analyzed with the pellet (insoluble fraction) by western blot. In immunoprecipitation (IP) experiments, nuclear extracts (1 mg) were incubated with antibodies overnight at 4°C and further processed using the Universal Magnetic Co-IP kit (Active Motif) according to the manufacturer's instructions. Antibodies used for immunoblotting were: 53BP1 (Abcam, Ab36823, 1 μg/ml), BRCA1 (Calbiochem, MS110, 5 μg/ml), BRG1 (Milipore, 07–478, 1:10000), DNA-PKcs (Abcam, clone 18–2, 2 μg/ml), γH2AX (Ser139; Millipore, clone JBW301, 1 μg/ml), Histone H2B (Abcam, Ab1790, 0.1 μg/ml), lamin B (Abcam, Ab16048, 60 ng/ml), NuMA (B1C11, 1:2, a gift from Dr Jeffrey Nickerson, UMass, Worcester, USA), PAR (Trevigen, 4336-APC-050, 1:1000), phospho-NuMA (Cell Signaling, 1:1000), SNF2h (Abcam, Ab3749, 1 μg/ml), RAD51 (Abcam, Ab63801, 1:1000), tubulin (Abcam, Ab3194, 1 μg/ml) and Williams Syndrome Transcription Factor (WSTF; Cell Signaling, 0.3 μg/ml). For IP: NuMA (Oncogene, clone Ab-2 or Bethyl Laboratories) and SNF2h (Abcam, clone 3.25(2)).
3
Antibody Characterization for Protein Study
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The antibodies used in this study were anti-Myc antibody (sc-40, Santa Cruz), anti-HA antibody (sc-57592, Santa Cruz), anti-HBO1 antibody (sc-13284, Santa Cruz), anti-DDB2 antibody (sc-81246, Santa Cruz), anti-XPC antibody (sc-30156, Santa Cruz), anti-TFIIH p89 antibody (sc-293, Santa Cruz), anti-CPD antibody (NMDND001, Cosmo Bio), anti-Orc2 antibody (sc-13238, Santa Cruz), anti-acetyl-Histone H4 antibody (#06-866 Millipore), anti-Histone H4 antibody (ab10158, Abcam), anti-acetyl-Histone H3K14 antibody (A-4023, EPIGENTEK), anti-Histone H3 (tri methyl K4) antibody (ab1012, Abcam), anti-Histone H3 antibody (39763, ACTIVE MOTIF), anti-γH2AX antibody (50-171-736, Upstate), anti-SNF2H antibody (ab3749, Abcam), anti-ACF1 antibody (NB100-61041, Novusbio), anti-CSB antibody (553C5a, BIO MATRIX RESEARCH), anti-UVsS-A antibody (H00057654-B01, Abnova) and anti-β-actin antibody (sc-47778, Santa Cruz). Phospho Ser50 and Ser53 HBO1 rabbit polyclonal antibodies were generated by immunization with a synthetic phosphopeptide (CSARLpSQSpSQD). To perform immunoprecipitation of GFP-SNF2H, anti-GFP mAb-Agarose (D153-8, MBL) was used.
4
Immunostaining of DNA Repair Factors
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This procedure was performed as described previously (19 (link)). Where indicated, immunostaining was preceded with in situ cell fractionation to reveal insoluble proteins (24 (link)). Fluorescent signals were imaged with a Zeiss CLSM710 using a 63× oil (NA = 1.4) objective. Repair foci were quantified using an automated routine in ImageJ (http://rsbweb.nih.gov/ij/ ) or by visual scoring. Antibodies used for immunostaining were: 53BP1 (Abcam, ab36823, 5 μg/ml), BRCA1 (Calbiochem, MS110, 0.3 μg/ml), cyclin B1 (Cell Signaling Technology, clone D5C10, 1:200), FLAG (Sigma, M2, 2 μg/ml), γH2AX (Milipore, JBW301, 3.3 μg/ml), Histone H4 acetylated (Milipore, 06–598, 5 μg/ml), Histone H4K20me (Abcam, ab9051, 5 μg/ml), Ki67 (Vector Laboratories, VP-K451, 1:1000), lamin B (Abcam, Ab16048, 2 μg/ml), NuMA (B1C11, 1:2), NuMA full-length (Abcam, ab36999, 1:100), NuMA proximal coiled-coil (Bethyl Laboratories, A301–510A, 4 μg/ml), NuMA distal C-terminal domain (Abcam, clone EP3976, 1:250), RAD51 (Abcam, ab63801, 1:1000), SNF2h (Abcam, ab3749, 15 μg/ml or clone 3.25(2), 4 μg/ml) and WSTF (Abcam, clone EP1704Y, 1:100). Note that for immunostaining with RAD51 antibodies, a permeabilization step with 0.5% triton X100 was performed after fixation with paraformaldehyde.
5
Protein Analysis via Western Blot
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Procedures for preparation of protein lysates and Western blots were followed as described [33] (link), [64] (link). For Western blot, 15 milligrams of whole-cell protein lysate was loaded per lane. Anti-V5 antibody (Pierce) was diluted 1∶5000 for use as the primary antibody. SWI2 antibody was obtained from Abcam (Ab3749). Protein purification prior to MS/MS analysis was performed using a slightly modified procedure [64] (link).
6
Western Blot Analysis of DNA Repair Proteins
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Protein lysates were prepared by scraping cells in 2 × sample buffer (125 mM Tris–HCl, pH 6.8, 20% glycerol, 10% 2-β-mercaptoethanol, 4% SDS, 0.01% bromophenol blue) and boiled at 98 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (0.45 µm, Merck Millipore). Subsequently, membranes were blocked in 2% BSA and incubated with primary antibodies and secondary antibodies conjugated with CF IRDye 680 and 770 (Sigma) for 1 h or overnight. Primary antibodies used were anti-XPA (sc-853, Santa Cruz Biotechnology), anti-SNF2H (ab3749, Abcam), anti-Tubulin (T6074, Sigma), anti-FANCD2 (nb100-316, Novus Biologicals), anti-GFP (ab290, Abcam), anti-SLX4 (NBP1-28680, Novus Biologicals), anti-ERCC1 (ab129267, Abcam), anti-H2B (07-371, Millipore), and anti-RPA70 (2267, Cell signaling). Secondary antibodies were visualized using the Odyssey CLx Infrared Imaging System (LI-COR Biosciences).
7
Genome-wide DNA Methylation Analysis
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Genomic DNA was treated using bisulfite, amplified (primers in Table S2 ), and subcloned. DNA from clones (>10 clones/amplicon) was sequenced as described elsewhere [15] (link).
For co-IP, 6×106 MEL or K562 cells were lysed (RIPA) and gently sonicated. 100 µg of precleared protein extract was incubated 3 hrs/4°C with anti-Ctcf (cat#07–729) or anti-Smarca5 (cat#07–624, Upstate) and next with proteinA/proteinG overnight. Control antibody: IgG, cat.# NI01, Calbiochem, 5∶100). Immunoprecipitates (IP) were washed with set of buffers. IPbuffer (0.02% SDS/2%Trion X-100/4 mM EDTA/40 mM Tris-HCl (pH = 8)/300 mM NaCl), WashI (0.1%SDS/1%Triton X-10/2 mM EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/50 mM NaCl), WashII (0.1% SDS/1% Triton X-10/1% EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/500 mM NaCl). IPs were resolved on SDS/PAGE, blotted, and immune-detected.
Chromatin immunoprecipitation (ChIP) [19] (link) lysates were controlled for DNA purity&quantity by Nanodrop ND-1000. Antibodies: Smarca5/Snf2h (cat.#ab3749, Abcam, 3 µg/IP), Ctcf (cat.#ab10571, Abcam, 2 µg/IP), RAD21 (cat.#ab992, Abcam, 2 µg/IP), and SMC1 (cat.# ab9262, Abcam, 2 µg/IP). Control IgG: cat.#NI01, Calbiochem, 5∶100.
For co-IP, 6×106 MEL or K562 cells were lysed (RIPA) and gently sonicated. 100 µg of precleared protein extract was incubated 3 hrs/4°C with anti-Ctcf (cat#07–729) or anti-Smarca5 (cat#07–624, Upstate) and next with proteinA/proteinG overnight. Control antibody: IgG, cat.# NI01, Calbiochem, 5∶100). Immunoprecipitates (IP) were washed with set of buffers. IPbuffer (0.02% SDS/2%Trion X-100/4 mM EDTA/40 mM Tris-HCl (pH = 8)/300 mM NaCl), WashI (0.1%SDS/1%Triton X-10/2 mM EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/50 mM NaCl), WashII (0.1% SDS/1% Triton X-10/1% EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/500 mM NaCl). IPs were resolved on SDS/PAGE, blotted, and immune-detected.
Chromatin immunoprecipitation (ChIP) [19] (link) lysates were controlled for DNA purity&quantity by Nanodrop ND-1000. Antibodies: Smarca5/Snf2h (cat.#ab3749, Abcam, 3 µg/IP), Ctcf (cat.#ab10571, Abcam, 2 µg/IP), RAD21 (cat.#ab992, Abcam, 2 µg/IP), and SMC1 (cat.# ab9262, Abcam, 2 µg/IP). Control IgG: cat.#NI01, Calbiochem, 5∶100.
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