Whole cell lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were resolved on 10–15% SDS-PAGE gels and electroblotted onto PVDF membrane. Membranes were blocked with 5% BSA or dried skimmed milk in TBS-T and incubated in primary antibodies in blocking buffer: anti-pRPA32-Ser33 (A300-246A Bethyl Laboratories), anti-phospho-γH2AX-Ser139 (Merck Millipore, 05–636), anti-pCHK1-Ser345 (Cell signalling #2348), anti-RNA polymerase II CTD repeat YSPTSPS (phospho-S5) (Abcam ab5401), anti-PUM2 (Bethyl Laboratories A300-202A), anti-RNASEH1 (SantaCruz Biotechnologies, H-4, sc-376,326), anti-β-actin (Abcam ab6276), anti-Vinculin (Merck Millipore, V9131), anti-GAPDH (Cell Signalling 14C10 #2118). Membranes were washed and incubated with HRP-conjugated secondary antibodies and blots developed with Luminata™ forte (Merck-Millipore) and imaged using iBright (Invitrogen).
Anti pchk1 ser345
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
The Anti-pCHK1 Ser345 is a lab equipment product that detects phosphorylated CHK1 at serine 345. It is used to analyze the activation of the cell cycle checkpoint protein CHK1 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Luminata forte, by Merck Group (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Anti pum2, by Fortis Life Sciences (1 mentions)
Anti phospho γ h2ax ser139, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti γh2ax, by GeneTex (1 mentions)
Anti plk1, by Abcam (1 mentions)
Anti wip1, by Abcam (1 mentions)
Anti aurora a, by Abcam (1 mentions)
Anti p21, by GeneTex (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti gapdh, by GeneTex (1 mentions)
Hrp goat anti rabbit igg antibody, by Vector Laboratories (1 mentions)
Anti chk2 pt68, by Cell Signaling Technology (1 mentions)
Anti phospho histone h2ax ser139, by Cell Signaling Technology (1 mentions)
Anti vinculin, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Fusion solo, by Vilber (1 mentions)
5 protocols using anti pchk1 ser345
1
Western Blot Analysis of DNA Damage Response
2
Protein Extraction and Western Blot Analysis
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Cells were harvested in TLB lysis buffer supplemented with the Complete C protease and phosphatase inhibitors mix (Roche, Mannheim, Germany). Quantification was made with Bradford ready to use reagent (Biorad, Hercules, CA). Total cell protein (10 μg) was separated by 12 % SDS- PAGE, transferred to nitrocellulose membrane (Biorad, Hercules, CA) and incubated with primary antibodies overnight at 4°C followed by incubation with goat-anti-mouse (Invitrogen, Carlsbad, CA, USA) or goat-anti-rabbit (Invitrogen, Carlsbad, CA, USA) HRP tagged secondary antibodies. Bands were visualized with Lumigen on Amersham Hyperfilm (GE Healthcare, Fairfield, CT, USA). Primary antibodies used are listed below: anti-WEE1 (NP_001137448.1) (Abcam, Cambridge, UK), anti-WIP1 (NP_003611.1) (Abcam, Cambridge, UK), anti-pCHK1 Ser345 (Cell Signaling, Boston MA, USA), anti- γH2AX (Genetex, Irvine, CA), anti-p21 (NP_000380.1) (Genetex, Irvine, CA), anti-MYT1 (NP_004526.1) (Genetex, Irvine, CA), anti-Aurora A (NP_003591.2) (Abcam, Cambridge, UK), anti-CDC25B (NP_001274445.1) (Genetex, Irvine, CA) and anti-PLK1 (NP_005021.2) (Abcam, Cambridge, UK); anti-GAPDH (NP_001243728.1) was used as loading control (Genetex, Irvine, CA).
3
Western Blot Analysis of DNA Damage Signaling
4
Plasma Protein Phosphorylation Analysis
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Plasma lysates were prepared and Western blot was performed as described previously [24 (link)]. Briefly, plasma from ten subjects from each group was prepared by lysing in radioimmunoprecipitation assay buffer (50 mmol/L Tris, pH7.3, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, and 0.1% SDS) with protease inhibitors, NaVO4 and NaF. One hundred micrograms of plasma lysates were resolved in 10% SDS-PAGE and then transferred to PVDF membranes. Equal loading and transfer of proteins was verified by Ponceau red staining of the membranes. Blots were incubated using the following antibodies: anti-pCHK1 (Ser345, 1:500 dilution, Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1:5000 dilution, Cell Signaling Technology, Danvers, MA, USA). Proteins were detected by chemiluminescence detection (Pierce ECL Western blot substrate, Thermo Fisher Scientific Inc., Rockford, IL, USA) and analyzed by FUSION Solo (Vilber Lourmat, Collégien, France).
5
HEK293 Cells Western Blot Analysis
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HEK293 cells (3 × 105) were seeded in 6-well plate and treatment with selected extracts was performed for 16 h unless indicated. For western blot analysis, cells were collected and homogenized in 1% Triton X-100 in PBS containing proteases and phosphatases inhibitors (Roche). After sonication, protein concentration was determined in all experiments by micro-BCA assay (Pierce), and 10–30 µg of total protein was loaded in 8–15% SDS-PAGE minigels (Bio-Rad Laboratories, Hercules, CA) prior transfer into PVDF membranes. Membranes were blocked using PBS, 0.1% Tween-20 (PBST) containing 5% milk for 60 min at room temperature and then probed overnight with primary antibodies in PBS, 0.02% Tween-20 (PBST) containing 5% skimmed milk. The following primary antibodies and dilutions were used: anti-GFP 1:1,000 (Santa Cruz, Cat. n° SC-9996), anti-HSP90 1:5,000 (Santa Cruz, Cat. n° SC-13119), anti-LC3 1:1,000 (Cell signaling, Cat. n° 3868), anti-p62 1:5,000 (Abcam, Cat. n° ab56416), Anti-pCHK1(Ser345) 1:1,000 (Cell Signaling, Cat. n° CST-2348). Bound antibodies were detected with peroxidase-coupled secondary antibodies incubated for 2 h at room temperature and the ECL system.
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