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Anti pchk1 s345

Manufactured by Cell Signaling Technology
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Anti-pCHK1-S345 is a laboratory antibody product that specifically recognizes the phosphorylated form of the Checkpoint kinase 1 (CHK1) protein at serine 345. This antibody can be used to detect and study the activation of CHK1 in various cellular and experimental systems.

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Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (4 mentions) Anti chk1, by Santa Cruz Biotechnology (3 mentions) Odyssey infrared scanner, by LI COR (2 mentions) Anti atr, by Santa Cruz Biotechnology (2 mentions) Anti chk1 g 4, by Santa Cruz Biotechnology (2 mentions) Anti chk1, by Cell Signaling Technology (2 mentions) Anti pchk2 t68, by Cell Signaling Technology (2 mentions) Anti pkap1 s824, by Fortis Life Sciences (2 mentions) Anti β actin, by Thermo Fisher Scientific (2 mentions) Anti ku70, by GeneTex (1 mentions) α tubulin t9026, by Merck Group (1 mentions) Anti p rpa32, by Fortis Life Sciences (1 mentions) Anti rpa32, by Santa Cruz Biotechnology (1 mentions) Anti γh2ax, by Merck Group (1 mentions) Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions) Anti skp2, by Cell Signaling Technology (1 mentions) Anti mouse alexa 488, by Jackson ImmunoResearch (1 mentions) Alexa 647 azide, by Thermo Fisher Scientific (1 mentions) Anti cdt1, by Cell Signaling Technology (1 mentions) Anti mcm2, by BD (1 mentions)

Close spelling variants of this product

PCHK1 S345 (37 citations) Rabbit anti-pCHK1 (s345, (3 citations)

9 protocols using anti pchk1 s345

1

SDS-PAGE and Western Blot Analysis of DNA Damage Response Proteins

Cited 1 time
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SDS-PAGE and western blot analysis were carried out according to the previously published protocol33 (link). Briefly, cells were collected by trypsinization and were washed in ice-cold PBS. Approximately 3 × 106 cells were lysed in ice-cold RIPA buffer (ThermoFisher Scientific) supplemented with Halttm phosphatase and protease inhibitor cocktails (ThermoFisher Scientific), as recommended by the manufacturer and processed for SDS-PAGE and subsequently for western blotting as described33 (link). The primary antibodies were: anti-RPA32 (mouse hybridoma cell line kindly provided by Dr. J. Hurwitz), anti-KU70 (529) (GeneTex, GTX77607), anti-ATR (Santa Cruz Biotechnology, sc-28901), anti-CHK1 (G-4) (Santa Cruz Biotechnology, sc-8408), anti-pCHK1-S345 (Cell Signaling Technology), anti-DNA-PKcs (Merck Millipore, PC127) and were used at 1:500 to 1:4000 dilutions. The secondary antibodies were anti-mouse IgG conjugated with IRDye680 or anti-rabbit-IgG conjugated with IRDye800 (Li-COR Biosciences, 92668020 and 92632211) at 1:15,000 dilution. Immunoblots were visualized by scanning the membranes in an Odyssey infrared scanner (Li-COR Biosciences). Digital images were processed using the brightness and contrast functions of the dedicated Odyssey software. Raw non-cropped scanned membranes are presented on Fig. S4A–C.
Mladenov E., Fan X., Paul-Konietzko K., Soni A, & Iliakis G. (2019). DNA-PKcs and ATM epistatically suppress DNA end resection and hyperactivation of ATR-dependent G2-checkpoint in S-phase irradiated cells. Scientific Reports, 9, 14597.
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2

Inhibition of USP1-UAF1 Complex in DNA Damage

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The following chemicals were used in this study: ML323 (S7529, an USP1-UAF1 inhibitor, 30 μM) (Selleckchem), and Dynabeads M-280 streptavidin (11206D) (Invitrogen). The following reagents were used in this study: Ultrapure 20X SSC (#15557044) (Invitrogen), hydrogen peroxide solution (H2O2) (H1009) (Sigma-Aldrich), formaldehyde (P2031), and formamide (FC1014) (Biosesang). The following antibodies were used: anti-PCNA (ab18197), anti-LIG1 (ab615) (Abcam), anti-ubiquityl-PCNA (Ub-PCNA) (Lys164) (13439S), anti-histone H3 (9175), anti-CHK1 (2360), anti-pCHK1 (S345) (2348), anti-γH2AX (9718) for immunoblotting (Cell signaling), anti-FLAG (F3165), anti-HA (H9658), anti-V5 (V8137), anti-Myc (05–724), anti-S tag (SAB2702227), α-tubulin (T9026) (Sigma-Aldrich), anti-RFC1 (NBP2-54960) (Novus Biologicals), anti-FEN1 (sc-13051) (Santa Cruz), anti-RPA32 (A300-244A), anti-pRPA32 (S4/S8) (A300-245A), anti-pRPA32 (S33) (A300-246A) (Bethyl), anti-S9.6 (ENH001) (Kerafast), and anti-γH2AX (05–036) for immunostaining (Millipore).
Kim S., Kim Y., Kim Y., Yoon S., Lee K.Y., Lee Y., Kang S., Myung K, & Oh C.K. (2023). PCNA Ser46-Leu47 residues are crucial in preserving genomic integrity. PLOS ONE, 18(5), e0285337.
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3

Antibody Sourcing for Cell Signaling

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The following antibodies were purchased from Cell Signaling Technologies: anti-pChk1 S345 (Cat# 2341), anti-Chk1 (Cat# 2345), anti-Cdt1 (Cat# 8064), and anti-Skp2 (Cat# 4313). Anti-HA used for immunoblotting was purchased from Roche (Cat# 11867423001). Anti-HA used for coimmunoprecipitation was purchased from Santa Cruz Biotechnology (Cat# SC-805). Anti-cyclin A was purchased from Santa Cruz Biotechnology (Cat# SC-596). Anti-MCM2 was purchased from BD Biosciences (San Jose, CA, Cat#610700). Anti-mouse Alexa 488 (Jackson ImmunoResearch) and Alexa 647-azide (Life Technologies) were used in flow cytometry analyses. Secondary antibodies for immunoblotting were purchased from Jackson ImmunoResearch.
Pozo P.N., Matson J.P., Cole Y., Kedziora K.M., Grant G.D., Temple B, & Cook J.G. (2018). Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication. Molecular Biology of the Cell, 29(25), 2989-3002.
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4

SDS-PAGE and Western Blot Analysis Protocol

Cited 3 times
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SDS-PAGE and Western blot analysis were carried out according to previously published protocols [13 (link),14 (link),16 (link)]. Briefly, cells were collected by trypsinization, washed in ice-cold PBS, and lysed in ice-cold RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Waltham, MA, USA), as recommended by the manufacturer. Protein lysates were resolved in SDS-PAGE gels and were transferred to nitrocellulose membrane for Western blot analysis. The primary antibodies were anti-Chk1 (G-4) (Santa Cruz Biotechnology, Heidelberg, Germany), anti-pChk1-S345, anti-pChk1-S296, anti-HSP27, anti-pHSP27-S82, anti-MK2 and anti-pMK2-T334 (all from Cell Signaling Technology, Leiden, The Netherlands), anti-Ku80 (GeneTex, Irvine, CA, USA), anti-Ku70 (N3H10) (GeneTex, Irvine, USA), and anti-GAPDH (MERCK, Darmstadt, Germany) at the corresponding dilutions (See Table S1 for details). The secondary antibodies were anti-mouse and anti-rabbit IgG conjugated with IRDye680 and IRDye800 (LI-COR Biosciences, Bad Homburg, Germany) at 1:10,000 dilution. Immunoblots were scanned on the Odyssey infrared scanner (LI-COR Biosciences, Bad Homburg, Germany). The RAW Western blot results are shown in Figures S6–S9.
Luo D., Mladenov E., Soni A., Stuschke M, & Iliakis G. (2023). The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G2-Checkpoint Activation in Cells Exposed to Ionizing Radiation. Cells, 12(10), 1387.
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5

Antibody Validation and Generation

Cited 3 times
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α-PCNA (P8825) and α-HIS (H1029) were purchased from Sigma; α-Cul2 (EPR3104) was purchased from Abcam; α-p97 (65278) was purchased from Progen Biotechnik, anti-P-Chk1 (S345) from Cell Signaling, γ-H2AX (4418-APC-020, Trevigen).
Affinity purified α-Cdc45, α-Psf2 (40 (link)), α-Mcm3 (41 (link)), α-LRR1 (S962D) and α-Cul2 (SA206) (4 (link)), and α-Mcm7 (6 ) were previously described. Xenopus Ufd1 antibody was a kind gift from Prof Stemmann’s lab (13 (link)).
Xenopus full-length p97, Ubxn7, Faf1, and GINS proteins were purified as described previously (6 , 13 (link)) and antibodies raised against such prepared antigens in sheep (p97, GINS, Ubxn7) or rabbit (Faf1). The resulting antibody sera were purified in-house against the purified antigen. The specificity of each new antibody is presented in Fig S7.
Tarcan Z., Poovathumkadavil D., Skagia A, & Gambus A. (2022). The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase. The Journal of Biological Chemistry, 298(8), 102234.
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6

Immunoblotting for DNA Damage Response

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Antibodies were obtained from the following commercial sources: anti-H2AxSer139 (Millipore; clone JBW301), anti-P53BP1Ser25 (Novus Biologicals; NB100-1803), anti-53BP1 (ThermoFisher Scientific; PA1-16565), anti-pChk1S345, anti-pChk2T68, anti-Chk1, anti-Chk2, anti-ATM (Cell Signaling Technology; 2348S, 2661S, 2G1D5, 1C12 and D2E2 respectively), anti-pATMS1981 (Rockland, NC9306342), anti-β-actin (Life Technologies, clone AC-15) and anti-vinculin (Abcam, clone ab18058).
Sears C.R., Cooney S.A., Chin-Sinex H., Mendonca M.S, & Turchi J.J. (2016). DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer. DNA repair, 40, 35-46.
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7

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.
Castella M., Jacquemont C., Thompson E.L., Yeo J.E., Cheung R.S., Huang J.W., Sobeck A., Hendrickson E.A, & Taniguchi T. (2015). FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2. PLoS Genetics, 11(10), e1005563.
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8

Chk1/Chk2 Signaling Pathway Analysis

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Cells were collected using a scraper, washed with PBS and resuspended in lysis buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA) containing protease and phosphatase inhibitor cocktail (Roche Molecular Biochemicals). The following antibodies with corresponding concentrations were used: anti-PAR (Trevigen, 1:1000), anti-Chk2 (EMD Millipore, 1:1000), anti-pChk2-T68 (Cell Signaling, 1:1000), anti-Chk1 (Santa Cruz, 1:750), anti-pChk1-S345 (Cell Signaling, 1:1000), anti-pKAP1-S824 (Bethyl Laboratories, 1:2000), anti-Cas9 (Cell Signaling Technology, 1:1000), anti-Clathrin Heavy Chain (BD Transduction Laboratories, 1:10 000) and anti-Tubulin (Sigma, 1:10 000).
Vriend L.E., Prakash R., Chen C.C., Vanoli F., Cavallo F., Zhang Y., Jasin M, & Krawczyk P.M. (2016). Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks. Nucleic Acids Research, 44(11), 5204-5217.
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9

DNA Damage Response Pathway Profiling

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The following primary antibodies were used: anti-β-Actin (Thermo Fisher Scientific; #MA1-140), anti-pDNA-PKcs-S2056 (Thermo Fisher Scientific; #PA5-78130), anti-DNA-PKcs (Bethyl Laboratories; #A300-516A), anti-pRPA2-S4/8 (Bethyl Laboratories; #A300-245A), anti-RAD51 (Merck Millipore; #PC130), anti-pKAP1-S824 (Bethyl Laboratories; #A300-767A), anti-pATM-S1981 (Cell Signaling Technology; #13050S), anti-KAP1 (Bethyl Laboratories; #A300-274A), anti-pCHK1-S345 (Cell Signaling Technology; #2341), anti-CHK1 (Santa Cruz Biotechnology; #sc-8408), anti-pH2AX-S139 (Bethyl Laboratories; A300-081A), anti-BRCA1 (Cell Signaling Technology; #9010), anti-CTIP (Bethyl Laboratories; #A300-488A), anti-BLM (Bethyl Laboratories; #A300-110A), anti-V5 (Thermo Fisher Scientific; #R960-25). The secondary antibodies used were Goat anti-Mouse IgG-HRP (Thermo Fisher Scientific; #NA931V) and Donkey anti-Rabbit IgG-HRP (Thermo Fisher Scientific; #NA934V).
Dibitetto D., Sanchi A., Sanford E.J., Lopes M., & Smolka M.B. (2021). Fork Slowing and Reversal as an Adaptive Response to Chronic ATR Inhibition.
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