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.
Anti fancd2
Manufactured by Novus Biologicals
Sourced in Germany
Anti-FANCD2 is a purified polyclonal antibody that recognizes the FANCD2 protein. FANCD2 is a key component of the Fanconi anemia DNA repair pathway. The antibody can be used to detect FANCD2 in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti fanca, by Fortis Life Sciences (3 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Axiovert lsm780 microscope, by Zeiss (2 mentions)
Anti pcna, by Abcam (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Aphidicolin, by Fujifilm (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti neil3, by Proteintech (1 mentions)
Anti fanci, by Santa Cruz Biotechnology (1 mentions)
Anti ruvbl2, by BD (1 mentions)
Anti γh2ax ser139, by Cell Signaling Technology (1 mentions)
Anti parp1, by Cell Signaling Technology (1 mentions)
Anti ha tag, by Cell Signaling Technology (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti h2b, by Abcam (1 mentions)
Anti blm, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti fancd2
1
Whole-Cell Lysis and Western Blot
2
Confocal Microscopy of FANCD2 in AICAR-treated Cells
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Confocal microscopy was performed as described previously 19 . Cells grown on coverslips and treated with AICAR for 24 h were fixed in 3.7% paraformaldehyde in PBS for 20 min, permeabilized with 0.2% Triton X‐100 in PBS for 10 min and blocked with 1% bovine serum albumin in PBS for 1 h. Fixed cells were incubated overnight with anti‐FANCD2 (Novus Biologicals), washed in PBS supplemented with 0.2% Tween 20 (PBST) and incubated with Alexa 488‐conjugated donkey anti‐rabbit antibody for 3 h. Finally, the cells were washed with PBST, stained with 4′,6‐diamidino‐2‐phenylindole (DAPI), mounted on glass slides and observed with a Zeiss Axiovert LSM780 microscope (Carl Zeiss, Oberkochen, Germany).
3
Western Blot Analysis of DNA Repair Proteins
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Cells were harvested and lysed in RIPA buffer containing protease inhibitor cocktail (Roche, Switzerland). The 20 μg of total protein were separated by SDS-PAGE and then transferred to PVDF membrane. After blocking in 10% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4° C. The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies, and visualized with ECL. The primary antibodies used were anti-Blm (1:1000, Invitrogen, USA), anti-Wrn (1:1000, Invitrogen), anti-Recql4 (1:1000, Invitrogen), anti-Timeless (1:1000, Invitrogen), anti-Ddx11 (1:1000, Invitrogen), anti-Pot1 (1:1000, Invitrogen), anti-Fancd2 (1:1000, Novus Biologicals, USA), anti-Terf1 (1:1000, Invitrogen), anti-Gins2 (1:500, Santa Cruz, USA), anti-Top2a (1:1000, Novus Biologicals), anti-Mcm7 (1:1000, Santa Cruz), anti-Mcm2 (1:1000, Abcam, UK), anti-PCNA (1:1000, Abcam), anti-H2B (1:1000, Abcam), anti-tubulin (1:5000, Proteintech, USA), anti-actin (1:1000, Santa Cruz).
4
Western Blot Analysis of DNA Repair Proteins
5
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 .
6
Protein Extraction and Western Blotting Protocol
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The tissue and cell proteins were isolated using radio immunoprecipitation assay (RIPA) lysis buffur (Epizyme Biotech) supplemented with protease inhibitor. The Bradford method (CWBIO) was used to determine the protein concentrations. Proteins were electrophoresed on Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, CWBIO), then transferred to polyvinylidene fluoride (PVDF) membranes, blocked, and incubated with primary anti-FANCE (Biobryt, 1:10000), anti-FANCD2 (1: 5000; Novus Biologicals), anti-GAPDH (1:10000, bioworld), anti-β-tubulin (1:10000, proteintech), anti-RAD51(1:5000, Abcam), anti-γH2AX (1:10000, Abcam), anti-cyclin dependent kinase 4 (CKD4) (1:5000, proteintech), anti-cyclin dependent kinase 6 (CDK6) (1:5000, proteintech), anti-cyclin D1 (1:5000, proteintech), anti-cyclin B1(1:5000, proteintech), anti-cyclin E(1:5000, proteintech) and anti-cyclin A2(1:5000, proteintech) for overnight at 4℃. Secondary antibodies were incubated for 2 h the next day. The protein bands on the membranes were detected using Enhanced Chemiluminescent (ECL, NCM Biotech).
7
Immunodetection of RAD51 and 53BP1 Foci
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For RAD51 and 53BP1 bodies immunodetection, unless otherwise stated, all dilutions were prepared in PBS and incubations were performed at room temperature with intervening washes in PBS. Cell fixation was carried out by incubation with 4% paraformaldehyde for 10 min followed by prechilled methanol for 5 min at −20 °C. This was followed by permeabilization in 0.2% TritonX-100 for 5 min and a quenching step using 0.1% sodium borohydride for 5 min. After blocking for 1 h in a solution containing 10% goat serum and 1% BSA, cells were incubated for 1 h with primary antibodies anti-RAD51 (1:5000, B-bridge International) and anti-cyclin A (1:400, BD Biosciences), anti-FANCD2 (1:1000, Novus) and anti-Geminin (1:2000, Abcam) or 53BP1 (1:1000, Novus) and anti-cyclin A diluted in 1% BSA. Secondary antibodies Alexa Fluor 568 goat anti-rabbit (1:1000, Thermo Fisher) and Alexa Fluor 488 goat anti-mouse (1:1000, Thermo Fisher) were diluted in 1% BSA and incubated for 1 h. Coverslips were mounted onto slides with ProLong® Gold Antifade Mountant with DAPI (Invitrogen life technology).
8
FAAP20 Expression in Tissue Lysates
9
FANCD2 Foci Quantification by Confocal Microscopy
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Confocal microscopy was performed as described previously 11 . Briefly, U2OS cells were grown on coverslips (18 mm in diameter; Marienfeld Superior) in 12‐well plates, fixed in 3.7% formaldehyde in PBS for 20 min, permeabilized with 0.2% Triton X‐100 in PBS for 20 min, and blocked with 1% bovine serum albumin in PBST. U2OS cells on coverslips were sequentially incubated with anti‐FANCD2 (Novus Biologicals) overnight and Alexa 488‐conjugated donkey anti‐rabbit secondary antibody for 2 h. After extensive washing with PBST [Phosphate Buffered Saline supplemented with 0.2% Tween‐20], cells were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI), mounted on glass slides, and observed under a Zeiss Axiovert LSM780 microscope (Carl Zeiss, Oberkochen, Germany). To count the number of foci, images were adjusted consistently by changing the gamma and white (brightness) parameters in the ZEN program (Carl Zeiss). After this adjustment, foci with pixel values of over 30 were counted.
10
Comprehensive Signaling Pathway Analysis
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Antibodies against Cdc42 (Cat# 2466S), phosphorylated-EGFR (p-EGFR, Cat# 2234), EGFR (Cat# 2232), p-ERK (Cat# 9102S), ERK (9102), p-AKT (S473, Cat# 4960), p-AKT (T308, Cat# 4056), AKT (Cat# 9272S), and STAT3 (Cat# 4904T) were obtained from Cell Signaling Technology. Antibodies against p-STAT3 (pS727, Cat# 612542) were purchased from BD Biosciences. Anti-Rac1 was obtained from Millipore (Cat# 05-389). Anti-FANCD2 was purchased from Novus Biologicals (Cat# NB100-361). Anti-β-actin antibody was obtained from Santa Cruz (Cat# sc-47778). MEK1 adenovirus was kindly provided by Dr. Jeffery Molkentin (Children'Hospital Medical Center, Cincinnati).
Bortezomib (Cat# S1013) and BVD523 (Cat# 869886-67-9) were obtained from Selleckchem. CASIN was obtained from Cayman Chemical Co. (Cat# 425399-05-9). For the in vitro experiments, CASIN was dissolved in DMSO to make the stock solution, followed by diluting it with the culture medium to a series of the testing solutions. For the in vivo experiments, CASIN was dissolved in cyclodextran. Melphalan was purchased from Sigma-Aldrich (Cat# 148-82-3). The protease inhibitor cocktail tablets were obtained from Roche Diagnostics GmbH (Ref# 11836170001). The phosphatase inhibitor cocktail was purchased from Goldbio (Cat# GB-450).
Bortezomib (Cat# S1013) and BVD523 (Cat# 869886-67-9) were obtained from Selleckchem. CASIN was obtained from Cayman Chemical Co. (Cat# 425399-05-9). For the in vitro experiments, CASIN was dissolved in DMSO to make the stock solution, followed by diluting it with the culture medium to a series of the testing solutions. For the in vivo experiments, CASIN was dissolved in cyclodextran. Melphalan was purchased from Sigma-Aldrich (Cat# 148-82-3). The protease inhibitor cocktail tablets were obtained from Roche Diagnostics GmbH (Ref# 11836170001). The phosphatase inhibitor cocktail was purchased from Goldbio (Cat# GB-450).
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