HeLa cells were transfected with the corresponding siRNAs and 48h after siRNA transfection, they were transfected with either the RNase H1-coding plasmid pEGFP-M27 or the control plasmid pEGFP-C1. After 72 h of siRNA transfection, cells were crosslinked and processed for ChIP using standard procedures with minor modifications as previously described [23 (link)]. Anti-FANCA (Bethyl Laboratories) or anti-γH2AX (clone JBW301; Upstate) previously conjugated with Dynabeads Protein A (Life Technologies) were used to immunoprecipitate chromatin.
Anti fanca
Manufactured by Fortis Life Sciences
Sourced in United States
Anti-FANCA is a laboratory product used for the detection and analysis of the FANCA protein. It serves as a research tool for investigating the FANCA gene and its associated cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti fancd2, by Novus Biologicals (3 mentions)
Anti chk1, by Santa Cruz Biotechnology (2 mentions)
Anti fanci, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Anti fancg, by Novus Biologicals (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Western lightening, by PerkinElmer (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Las4000 imaging system, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Fitc conjugated anti mouse iga, by BD (1 mentions)
Anti h2a, by Cell Signaling Technology (1 mentions)
Anti h2ax s139p, by Cell Signaling Technology (1 mentions)
Anti rad52, by Santa Cruz Biotechnology (1 mentions)
9 protocols using anti fanca
1
ChIP Assay for FANCA and γH2AX
2
Immunoblotting of Fanconi Anemia Proteins
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Cells were lysed with NETN300 buffer (300 mM NaCl, 0.2 mM EDTA, 50 mM Tris [pH 7.5], 1% NP40) supplemented with protease inhibitor cocktail (Roche). Cellular lysates were resolved by NuPAGE (Invitrogen) gels and transferred onto polyvinylidene fluoride (PVDF) membrane (EMD Millipore) followed by immunoblotting using antibodies as indicated: anti-FANCA (Bethyl), anti-FAAP20 (Sigma Atlas) and anti-Tubulin (Sigma). Signals were detected by either enhanced chemiluminescence method (Western Lightening, Perkin Elmer) or LAS-4000 Imaging system (GE Healthcare Life Sciences).
3
Immunoblotting analysis of DNA repair proteins
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Whole cell lysis and immunoblotting were performed as described63 (link),68 (link). Antibodies against FANCM and FAAP24, and antibody against BRCA2 were kindly provided by Dr. Weidong Wang38 (link) and Dr. Jun Huang66 (link), respectively. Antibodies against Mre11 and CtIP were used previously10 (link),63 (link),69 (link). Commercial antibodies used were: Anti-FANCA (Bethyl Laboratories A301-980A,1:1000), anti-FANCD2 (Bethyl Laboratories A302-174A, 1:1000), anti-MHF1 (Abcam, ab169385, 1:1000), anti-Rad51 (Santa Cruz Biotechnology, sc-398587, 1:500), anti-H2AX-S139p (Cell Signaling, #2577, 1:1000), anti-H2A (Cell signaling, #2578, 1:1000), anti-FLAG (Sigma, F1804, 1:2000), anti-Rad52 (Santa Cruz Biotechnology, sc-365341, 1:1000), anti-Ras (Santa Cruz Biotechnology, sc-520, 1:1000), anti-β-actin (Sigma, A5441, 1:2000), anti-Chk1 (Santa Cruz Biotechnology, sc-8408, 1:500), anti-Chk1-S345p (Cell Signaling,#2348, 1:500), anti-RPA32 (Bethyl Laboratories A300-244A, 1:1000), BRCA1 (Bethyl Laboratories, A300–000A, 1:500) and FITC-conjugated anti-mouse IgA (BD, 559354).
4
Whole-Cell Lysis and Western Blot
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To prepare whole-cell extracts, cells were washed once with ice-cold PBS and lysed with SDS lysis buffer (100 Mm pH 6.8 Tris–HCl, 20% glycerol, 1% SDS). Equal amounts of samples were subjected to 3–8% Tris-acetate gel (for FANCD2-ubiquitination detection) or 4–12% Bis–tris gel (Thermo Fisher Scientific). The protein was transferred to a PVDF membrane. Membranes were blocked in 2% BSA in TBS-T for 1 h and then incubated overnight at 4°C with the following antibodies: anti-FLAG M2 antibody (Sigma-Aldrich #F1804), anti-HA-tag (Cell Signaling Technology #3724), anti-GFP (Cell Signaling Technology #2555), anti-beta-actin (Cell Signaling Technology #4967), anti-GAPDH (Cell Signaling Technology #2118), anti-NEIL3 (Proteintech, 11621-1-AP), anti-FANCA (Bethyl Laboratories #A301-980), anti-FANCD2 (Novus Biologicals #NB100-182), anti-FANCI (Santa Cruz Biotechnology #sc-271316), anti-γH2A.X (Ser139) (Cell Signaling Technology #2577), anti-PARP1 (Cell Signaling Technology #9542), anti-PARP3 (Novus Biologicals #NBP1-31415), anti-MCM7 (Cell Signaling Technology #3735), anti-RUVBL1 (Abcam #ab51500) and anti-RUVBL2 (BD Biosciences #612482). Proteins were detected using a chemiluminescence system with a horseradish peroxidase conjugated secondary antibody.
5
Immunofluorescence and Western Blotting
6
DNA Damage Response Pathway Assay
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The following antibodies were obtained from commercial sources: anti-DNA:RNA (S9.6, Kerafast); anti-DDDDK-tag (MBL); anti-FLAG (M2, Sigma-Aldrich); anti-FLAG M2 magnetic beads (Sigma); normal mouse IgG (Santa Cruz); anti-FANCA (Bethyl); anti-FANCD2 (Novus); anti-PCNA (PC10, Santa Cruz); anti-γH2AX (JBW301, Millipore); anti-RPA (9H8, Abcam); anti-a-tubulin (T5168, Sigma). Aphidicolin (Wako), mitomycin C (MMC) (Kyowa Hakko Kirin), or cordycepin (Wako) were used at the indicated concentrations. Primers and siRNA oligos are summarized in Supplemental Table S1 .
7
AMPK and Fanconi Anemia Protein Interaction
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HEK 293T cells were transfected with pcDNA3-V5-PRKAA1 and pcDNA3-HA-FANCG or pcDNA3-HA-FANCA or the pcDNA3 empty vector using the Effectene Transfection Reagent (Qiagen, Valencia, CA, USA). The cells were treated 1 day later with 200 ng/mL MMC for 16 h and then lysed in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.3% Igepal CA-630, 0.2% Triton X-100, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors). Co-immunoprecipitation was performed as described previously [17 (link)]. Briefly, cell lysates were precleared with 10 μL protein A-agarose beads (Invitrogen, Carlsbad, CA, USA) and incubated with anti-HA antibody-conjugated agarose beads (Sigma, St. Louis, MO, USA), anti-V5 antibody-conjugated agarose beads (Sigma), or an anti-FANCA antibody (A301-980A, Bethyl Laboratories, Montgomery, TX, USA) with protein A-agarose beads for 18 h at 4°C. The beads were washed with lysis buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Co-immunoprecipitated proteins were detected by immunoblotting with anti-V5 (Invitrogen), anti-FANCA (Bethyl Laboratories), anti-FANCG (Novus Biologicals, Littleton, CO, USA), or anti-AMPKα (Cell Signaling Technology, Danvers, MA, USA) antibodies.
8
Immunoblotting with Diverse DNA Damage Proteins
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The following primary antibodies were used: anti-FANCD2 (Abcam, ab2187), anti-FANCA (Bethyl, A301-980A), anti-Vinculin (Sigma, V9131), anti-ATR (Santa Cruz, sc-1887), anti-FANCI (Santa Cruz, sc-271316), anti-Actin (Santa Cruz, sc-1616), anti-BRCA1 (Santa Cruz, sc-6954), anti-CHK1 (Santa Cruz, sc-8408), anti-pCHK1 S345 (Cell Signalling, 23415), anti-FLAG M2 (Sigma, F1804), anti-γH2AX (Upstate, JBW3001), anti-CyclinA (Abcam, ab16726), anti-MYC tag 9E10 (Upstate, 05–419), anti-TRF1 (Abcam, ab10579), anti-RAD51 (CosmoBio, BAM-70-001-EX), anti-FANCJ (Sigma, B1312), anti-BRCA2 (Calbiochem, OP95), anti-ABRAXAS (Bethyl, A302-180A), anti-RAP80 (Bethyl, A300-763A). Anti-FANCG [67 (link)], FANCC [68 (link)], FANCE [69 (link)], FANCF [70 (link)] and FANCD2 pT691 [71 (link)] were gifts from Dr. Alan D’Andrea (Dana-Farber Cancer Institute, Boston). Anti-USP1 (C-ter) [38 (link)] was a gift from Dr. Tony Huang. Anti-PALB2 [72 (link)] was a gift from Drs. David Livingston and Bing Xia. Anti-FANCL and anti-FANCM were gifts from Dr. Weidong Wang.
9
Western Blot Analysis of DNA Repair Proteins
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To prepare whole-cell extracts, cells were washed once with ice-cold phosphate-buffered saline and lysed with radioimmunoprecipitation buffer (Cell Signaling Technology, Danvers, MA, USA). Equal amounts of samples were subjected to 3À8% Tris-acetate gel (Thermo Fisher Scientific). The protein was transferred to a PVDF membrane. Membranes were blocked in 2% BSA in TBS-T for 1 hour and then incubated overnight at 4˚C with the following antibodies: anti-Myc-tag (Cell Signaling Technology, 2276), anti-beta-actin (Cell Signaling Technology, 4967), anti-FANCA (Bethyl Laboratories, A301-980), anti-FANCC (Abcam, ab54631), anti-FANCG (Novus Biologicals, NB100-2566), and anti-FANCD2 (Novus Biologicals, NB100-182). Proteins were detected using a chemiluminescence system with a horseradish peroxidase-conjugated secondary antibody. For FANCD2 monoubiquitination, cells were treated with 4 mmol/L hydroxyurea (HU) for 6 hours before protein extraction.
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