Primary antibodies used for western blot experiments: phospho-p53 Ser15, cleaved Caspase-3 and cleaved PARP (Cell Signaling, Leiden, The Netherlands), MAPK15 (custom preparation), p53 (Santa Cruz Biotechnology), ACTB (Sigma Aldrich), CDKN1A/p21 (Epitomics, Burlingame, CA), LC3B (Novus Biologicals, Cambridge, UK), SQSTM1/p62 (BD Biosciences, Milan, Italy), GADD45a (Cell Signaling), phospho-ATR (Cell Signaling), phospho-CHK2 (Cell Signaling), PCNA (Cell Signaling). Primary antibodies used for immunofluorescence experiments: γH2A.X (Cell Signaling), 53BP1 (Novus Biologicals) LC3B (MBL, Woburn, MA). Primary antibody used for immunohistochemistry experiments: MAPK15 (custom preparation). Secondary antibodies used for western blot experiments: HRP-conjugated anti-Mouse IgG and HRP-conjugated anti-Rabbit IgG (Santa Cruz Biotechnology). Secondary antibodies used for immunofluorescence experiments: AlexaFluor488-conjugated anti-Rabbit IgG and AlexaFluor555-conjugated anti-Mouse IgG (Life Technologies).
Phospho chk2
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Chk2 is a primary antibody that recognizes Chk2 protein phosphorylated at Thr68. Chk2 is a serine/threonine protein kinase that is activated in response to DNA damage and functions as a key regulator of cell cycle checkpoints.
Automatically generated - may contain errors
Lab products found in correlation
Phospho chk1, by Cell Signaling Technology (13 mentions)
Phospho p53, by Cell Signaling Technology (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (8 mentions)
Phospho atr, by Cell Signaling Technology (6 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
γh2ax, by Cell Signaling Technology (5 mentions)
Cleaved parp, by Cell Signaling Technology (5 mentions)
Phospho atm, by Cell Signaling Technology (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (4 mentions)
Phospho histone h2a x, by Cell Signaling Technology (3 mentions)
Cyclin d1, by Cell Signaling Technology (3 mentions)
Total chk2, by Cell Signaling Technology (3 mentions)
Cleaved caspase 9, by Cell Signaling Technology (3 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Species specific horseradish peroxidase conjugated secondary antibodies, by Santa Cruz Biotechnology (2 mentions)
Total caspase 3, by Cell Signaling Technology (2 mentions)
Total caspase 9, by Cell Signaling Technology (2 mentions)
Phospho chk1 ser296, by Cell Signaling Technology (2 mentions)
29 protocols using phospho chk2
1
Antibody Characterization for Cell Signaling
2
Immunoblot Analysis of Cellular Stress Response
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Cells were lysed in RIPA Buffer (Boston BioProducts #BP-115), supplemented with protease inhibitor cocktail tablets (Roche, complete mini, #11 836 153 001), and 5 mM NaF. Protein was quantified via Bio-RAD Protein Assay Dye Reagent Concentrate (BIO-RAD, #500-0006) on the Beckman Coulter DU640 Spectrophotometer. Proteins were separated by PAGE and electo-transferred to PVDF membranes (Pall Corporation, BioTrace PVDF 0.45 um, P/N 66543). Membranes were blocked in 5% milk tris-buffered saline with tween 20 (0.1%; TBS-T). Primary antibodies were incubated overnight on a rocker in 5% bovine serum albumin in TBS-T at 1:1000 at 4°C. Membranes were washed the following day 3×, 5′, TBS-T, and incubated with either mouse or rabbit horseradish peroxidase secondary (Cell Signaling #7076S and #7074) for 2 hours room temperature. Proteins were detected via film following the Thermo Scientific's SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, #34087) or Thermo Scientific's SuperSignal West Femto Maximum Sensitivity Substrate (#34095) protocol. The following antibodies were purchased from Cell Signaling Technology: B-Actin (#3700), BiP (#3177), CHOP (#2895), ATF-4 (#11815), Rad51 (#8875), Phospho-Histone H2A.X (#2577), H2A.X (#2595), ATM (#2873), Phospho-ATM (#13050), CHK2 (#6334), Phospho-Chk2 (#2661).
3
Western Blot Analysis of Cell Signaling
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Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (Sigma) and proteins were migrated on 10% or 12.5% SDS-PAGE gels and transferred to nitrocellulose or polyvinyldifluoride membranes. The membranes were blocked in 5% low fat milk for 1 h, and probed with the relevant primary antibodies overnight at 4 °C. The primary antibodies γ-H2AX, cleaved caspase-3, cleaved caspase-8, caspase-9, phospho-CHK1, phospho-CHK2, cyclin-D1, CDK6, CDK4, p21, ERK1/2, phospho-ERK1/2, p38 MAPK, phospho-p38 MAPK, JNK, phosphor-JNK, c-Jun, phospho-c-Jun and β-actin were obtained from Cell Signaling Technology (CST) (Beverly, MA, USA). Membranes were then probed with the appropriate secondary antibodies (CST) for 1 h at RT. The membranes were revealed using the Odyssey two-color infrared laser imaging system (LICOR, Lincoln, NE, USA). The ERK1/2 inhibitor U0126 and JNK inhibitor SP600125 were from Selleckchem.
4
Western Blot Analysis of DNA Damage Signaling
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Cells were collected, centrifuged, and re-suspended in radioimmunoprecipitation assay (RIPA) buffer plus protease inhibitors. The cells were then incubated in RIPA for 15 min at 4°C and centrifuged at 4°C for 15 min. Protein concentrations were determined using a Bradford protein assay. The samples were then resuspended in 4× lithium dodecyl sulfate sample buffer and denatured. Protein samples were electrophoresed on 4–20% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and then transferred to nitrocellulose membranes in transfer buffer (20% methanol, 11% glycine, and 3.4% Tris) at 100 V. The membranes were blocked with 5% milk in Tris-buffered saline—0.1% Tween (TBST) at room temperature for 45 min. The membranes were then incubated overnight in 5% milk in TBST at 4°C with primary antibodies. Membranes were subsequently washed with TBST and exposed to horse radish peroxidase-conjugated secondary antibodies in 5% milk in TBST at room temperature for 1 hr. The membranes were developed and enhanced using an enhanced chemiluminescent kit.
The primary antibodies used were: CtIP (14-1; (31 (link))), beta-actin (NB600-501SS, Novus), tubulin (3708-100, BioVision), Exo1 (ab95068, Abcam), DNA2 (PA5-23691, Invitrogen), phospho-Chk2 (2197S, Cell Signaling), phospho-p53 (9284, Cell Signaling) and GAPDH (sc-47724, Santa Cruz).
The primary antibodies used were: CtIP (14-1; (31 (link))), beta-actin (NB600-501SS, Novus), tubulin (3708-100, BioVision), Exo1 (ab95068, Abcam), DNA2 (PA5-23691, Invitrogen), phospho-Chk2 (2197S, Cell Signaling), phospho-p53 (9284, Cell Signaling) and GAPDH (sc-47724, Santa Cruz).
5
Immunoblotting Analysis of Apoptotic Markers
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Protein was analyzed by SDS-PAGE as previously described (16 (link)). The following primary antibodies from Cell Signaling Technology were used at manufacturer-recommended dilutions for immunoblotting: cleaved caspase-3 (#9661), total caspase 3 (#9668), cleaved caspase-9 (#9501), total caspase-9 (#9502), phospho-Chk1 (Ser296; #2349), total Chk1 (#2360), phos-pho-Chk2 (Thr68; #2661), total Chk2 (2662), and γH2AX (#9718). β-Actin (Santa Cruz Biotechnology, catalog #sc-47778) was included as a loading control. Species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used at 1:20,000 dilution.
6
Immunoblotting Analysis of Apoptotic Markers
7
Western Blot Analysis of Cell Signaling
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Following the experimental treatment, Western blot analysis were performed as previously described (6 (link)). Immunoblot analyses were carried out on lysates extracted from cells or tumors. Protein concentration was determined, and equal amounts of total proteins were separated on SDS-PAGE. Antibodies used included total (SCT #2602) and phospho-Pak1(SC #2606), Mek (SCT#9121), phospho-Mek pSer298(SCT#9128), Erk (SCT#9102), phospho-Erk1/2 (pThr202/pTyr204) (SCT#9101), phospho-ATM, phospho-ATR, phospho-Chk1, phospho-Chk2, phospho-p53, p21Cip SCT DNA damage sampler kit, #9947), cyclin B(SCT#4128), cyclin D1(SCT#2978), GAPDH (SCT#4138) were from Cell Signaling Technology.
8
UV-Induced DNA Damage Response Signaling
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For immunoblotting, cells were pretreated with 20 μM of FSK for 30 minutes before irradiation with UV at a dose of 20 J/m2. Harvested cells were resuspended in 100 μl 1× lysis buffer [20 mM Tris-HCl (pH 6.8), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1× protease inhibitor cocktail, and 10% Triton X-100] then sonicated (SONICS). Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis using 10% polyacrylamide gels and transferred to nitrocellulose blotting membranes using electrophoresis chambers (Bio-Rad). Blocked membranes were incubated with antibodies to CDT1 (Bethyl Laboratories), cyclin B1, CHK1 and CHK2 (Santa Cruz Biotechnology), phospho-CREB, CREB, phospho-CHK1, phospho-CHK2 and GAPDH (Cell Signaling Technology).
9
Comprehensive Molecular Profiling Protocol
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Antibodies against the following proteins were used: phospho-ATM (Ser 1981) (#5883), ataxiatelangiectasia mutated, ATM (#2873), phospho-AMPK (Thr172) (#2535), AMPKα1 (#2795), phospho-AMPK substrates (#5759), ATG7 (#8558), cleaved caspase 3 (#9664), phospho-CHK1 (Ser 345, 133D3) (#2348), CHK1 (2G1D5) (#2360), phospho-CHK2 (Thr 68) (#2197), CHK2 (#2662), p21 Waf1/CIP1 (12D1) (#2947), p27 Kip 1 (D69C12) XP (#3686), phospho 4EBP1 (Thr 37/46) (#9459), 4EBP1 (#9452), phospho-p70S6 kinase (Thr389) (#9205), p70S6 kinase (#9202), γH2AX (Ser139) (#9718), H2A.X (D17A3) XP (#7631) and phospho-MTOR (Ser2448) (#2971) all from Cell Signaling Technology); Ki67 (30-9) (#790-4286) from Roche; PARP1 (C-2-10) (#BML-SA249-0050) from Enzo Life Sciences; p62 (#610832) from BD Biosciences; ATG5 (#0262-100/ATG5-7C6) from Nanotools; ACTIN β (#NB600-501) from Novus Biologicals; LC3 (#M152-3) from MBL; phospho-p62 (Ser403) (#MABC186) from Merck Millipore; p16 (#805-4713) from Ventana; horseradish peroxidase-conjugated anti-rabbit (#111-035-003) and horseradish peroxidase (HRP)-conjugated anti-mouse (#115-035-174) from Jackson ImmunoResearch; and anti-mouse (#A11001) and anti-rabbit Alexa Fluor 488 (#A11008) from Invitrogen.
10
Western Blot Analysis of DNA Damage Response
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