Whole cell lysate were immunoblotted as previously described [5 (link)]. The primary antibodies used for immunoblotting were described as follows. The rabbit antibody against human BMCC1 was generated by MBL (Nagoya, Japan) [5 (link)]. The Anti-E2F1 (#3742), anti-phospho-ATM (Ser1981) (#4526), anti-phospho-Chk2 (Thr68) (#2661), anti-phospho-H2A.X (Ser139) (#9718), anti-phospho-FOXO3a (Thr32) (#9464), anti-FOXO3a (#2497), anti-PARP (#9542, Fig. 5 a and b) and anti-caspase-9 (#9502), anti-Bim (#4582), HRP-conjugated anti-Rabbit (#7074) and anti-Mouse (#7076) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-Human phospho-E2F1 (pS364) was purchased from ROCKLAND (Limerick, PA) and anti-p73 (ab-3) was obtained from Millipore (Billerica, MA), whereas anti-ATM (sc-23,921), anti-PARP-1 (sc-8007), and anti-Actin (sc-8432) were bought from Santa Cruz Biotechnology (Dallas, TX).
Anti phospho h2ax ser139
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-H2AX (Ser139) is a lab equipment product manufactured by Cell Signaling Technology. It is an antibody that specifically detects phosphorylation of histone H2AX at serine 139.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho chk2 thr68, by Cell Signaling Technology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti phospho chk1 ser345, by Cell Signaling Technology (2 mentions)
Anti actin sc 8432, by Santa Cruz Biotechnology (1 mentions)
Anti foxo3a, by Cell Signaling Technology (1 mentions)
Anti bim, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Enhanced chemifluorescence detection system, by Cytiva (1 mentions)
Anti phospho histone h3 ser10, by Cell Signaling Technology (1 mentions)
Rabbit anti rad51, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay systems, by Beyotime (1 mentions)
Anti nfbd1, by Abcam (1 mentions)
Anti phospho atm s1981, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Odyssey v3, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti clk1, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti phospho h2ax ser139
1
Immunoblotting Analysis of Cell Signaling Proteins
2
DNA Damage Response Protein Analysis
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The antibodies used in this study were: mouse and rabbit anti-NFBD1 (Abcam, London, UK); anti-ATM (phospho S1981) (Abcam); anti-DNA PKcs (phospho T2609) (Abcam); anti-phospho-H2AX (Ser139) (Cell Signaling Technology, Danvers, MA, USA); anti-phospho-histone H3 (Ser10) (Cell Signalling Technology); rabbit anti-Rad51 (Santa Cruz Biotechnology, Dallas, TX, USA). Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). Protein concentrations were determined using the BCA protein assay systems (Beyotime Institute of Biotechnology, Nantong, China). Proteins were fractionated in SDS–polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and western blotting were performed by using the appropriate antibody. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA) and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences, Piscataway, NJ, USA) under the room temperature.
3
Western Blot Analysis of Phosphorylated Proteins
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For Western blotting, HeLa cells were lysed with RIPA buffer (see Section 2.4 ), and 50 μg of protein was mixed with Laemmli sample buffer [13 (link)] and resolved in 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membrane (Millipore, Billerica, MA, USA), and membranes were blocked in TBS-T with 5% milk, for 1 h at room temperature. Then, membranes were incubated with one of the following primary antibodies diluted in TBS-T: anti-phospho-Chk1 Ser345 (Cat. number 2341), anti-phospho-Chk2 Thr-68 (Cat. number 2661), or anti-phospho-H2AX Ser139 (Cat. number 9718) polyclonal/monoclonal antibodies from Cell Signaling (Danvers, MA, USA) or an anti-α-Tubulin polyclonal/monoclonal antibody (B-7, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with appropriate species-specific IRDye (Infrared Dye) secondary antibodies (680 or 800 nm, diluted to 1 : 15000 in TBS-T) for 1 h and visualized and analyzed (by band density quantification) using an Odyssey Infrared Imaging System and the Odyssey V3.0 software (both from Li-COR Biosciences, Lincoln, NE, USA).
4
Comprehensive Antibody Panel for Cell Death Analysis
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Anti-PARP1 (100573), anti-BAX (109683), anti-BCL2 (100064), anti-cytochrome c
(108585), anti-COX-IV (101499), anti-a-tubulin (112141), anti-RIPK1 (111074),
anti-RIPK3 (107574), and anti-histone H3 (122148) were purchased from GeneTex
(ICON-Genetex Inc., Taipei, Taiwan); Anti-caspase-9 (ab115161) and
anti-caspase-3 (ab90437) were purchased from Abcam. Anti-MLKL (MACB604) was
purchased from Millipore. Anti-AIF (#4642), anti-Drp1 (#5391), anti-Drp1 Ser616
(#3455) and anti-phospho-H2AX (Ser139) (#9718) were purchased from Cell
Signaling Technology (MA, USA). Anti-b-actin (A5411) was purchased from
Sigma.
(108585), anti-COX-IV (101499), anti-a-tubulin (112141), anti-RIPK1 (111074),
anti-RIPK3 (107574), and anti-histone H3 (122148) were purchased from GeneTex
(ICON-Genetex Inc., Taipei, Taiwan); Anti-caspase-9 (ab115161) and
anti-caspase-3 (ab90437) were purchased from Abcam. Anti-MLKL (MACB604) was
purchased from Millipore. Anti-AIF (#4642), anti-Drp1 (#5391), anti-Drp1 Ser616
(#3455) and anti-phospho-H2AX (Ser139) (#9718) were purchased from Cell
Signaling Technology (MA, USA). Anti-b-actin (A5411) was purchased from
Sigma.
5
Antibody Validation for Immunoassays
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The following antibodies were used for immunoblotting, immunocytochemistry and immunoprecipitation: anti-SRPK1 (611072, BD Biosciences), anti-Hsp90 (610418, BD Biosciences), anti-Tip60 (DR1041, Calbiochem), anti-CLK1 (ab74044, Abcam), anti-myc (#2276, Cell Signalling Tech.), anti-Flag (#14793, Cell Signaling Tech.), anti-PARP (#9542, Cell Signaling Tech.), anti-phospho-H2AX (Ser139) (#9718, Cell Signaling Tech.), anti-β-actin (sc-47778, Santa Cruz), anti-GAPDH (#5174, Cell Signaling Tech.), anti-14-3-3β (sc-59419, Santa Cruz) and anti-Lamin A (sc-6214, Santa Cruz). The antibodies were diluted 1:100 for immunofluorescence and immunoblotting. For Monoclonal antibody MAb104, hybridoma cells (ATCC® CRL-2067™) were raised in 10% FBS-containing RPMI, and the conditioned medium was collected every two days, which could be directly used for immunoblotting.
6
Lentiviral Knockdown of DNA Repair Genes
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The following lentiviral constructs encoding shRNA-specific sequences targeting PRKDC (TRCN0000195491 and TRCN0000006255) and RPA2 (TRCN0000231924 and TRCN0000231921) transcripts on pLKO.1puro or pLKO_005 backbones were from the GPP library. Lentivirus preparations and infections were performed as described previously. Cells were lysed for immunoblot analysis at 6-, 10-, and 20-days post-infection. Whole-cell lysates, electrophoresis and immunoblotting were carried out as described previously (33) with the following antibodies and dilutions: anti-DNA-PKcs (#4602S, Cell Signaling), 1:1000; anti phospho-DNA-PKcs Ser2056 (#68716S, Cell Signaling), 1:1000; anti-RPA32/RPA2 (#52448S, Cell Signaling), 1:1000; anti-phospho-RPA32/RPA2 Ser8 (E5A2F, #54762S, Cell Signaling), 1:1000, anti-phospho-H2AX Ser139 (#9718S, Cell Signaling), 1:200; PARP (9532, Cell Signaling), 1:1000; Cleaved PARP (#9541S, Cell Signaling), 1:1000; anti-CHK1 (#8408, Cell Signaling), 1:1000; anti-phospho-CHK1 Ser345 #2348, Cell Signaling), 1:1000; and anti-GAPDH (G8795, Sigma), 1:5000.
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