HCT116 cells and their knockout derivatives or transfectants were cultured in each well of 6-well plates with normal medium until they reached 70%-80% confluence. Cell culture medium was replaced to medium with 0.1% FBS for 24 hours. Then the cells were treated with Chel A with indicated concentrations and time periods. following by being washed with ice-cold PBS, and then extracted with cell lysis buffer (10 mM Tris-HCl, pH 7.4, 1% SDS, 1mM Na3VO4, and proteasome inhibitor). The cell extracts were subjected to Western blotting with each of the antibodies for determination of PARP, Cleaved PARP, Caspase 3, Cleaved Caspase 3, p53, p-p53 Ser20, p-p53 Ser15, p-MDM2 Ser166, Chk2, p-Chk2 Thr68, p-Chk1 Ser345, ATR, p-ATR Ser428(Cell Signaling, Beverly, MA, USA), Catalase (Calbiochem, EMD Biosciences, Inc., La Jolla, CA, USA), MDM2, β-Actin (Sigma, St. Louis, MO, USA), as indicated. The protein bands specifically bound to the primary antibodies were detected using an alkaline phosphatase-linked secondary antibody and an ECF (enhanced chemifluorescence) Western blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA), as previously described [34 (link), 36 , 37 (link)].
P atr ser428
Manufactured by Cell Signaling Technology
Sourced in United States
The P-ATR Ser428 is an antibody that detects phosphorylation of the ATR (Ataxia Telangiectasia and Rad3-related) protein at serine 428. It can be used in techniques such as western blotting and immunohistochemistry to study the activation of the ATR signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P chk1 ser345, by Cell Signaling Technology (3 mentions)
P chk2 thr68, by Cell Signaling Technology (3 mentions)
P p53 ser20, by Cell Signaling Technology (2 mentions)
P p53 ser15, by Cell Signaling Technology (2 mentions)
P atm ser1981, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
P mdm2 ser166, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Alexa fluor 488 green conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 red goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Merck Group (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Patm ser1981, by Merck Group (1 mentions)
Horseradish peroxidase hrp, by Thermo Fisher Scientific (1 mentions)
Dylight 550, by Thermo Fisher Scientific (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
γh2ax, by Merck Group (1 mentions)
Alexa 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Nedd9, by Abcam (1 mentions)
6 protocols using p atr ser428
1
Chel A-Induced Cell Signaling Pathways
2
Immunofluorescence Microscopy of ATR and pATR
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Immunostaining was conducted as follows: MNNG/HOS and 143B were grown at a density of 2 × 104 cells/ml in 12-well plates for 2 days, fixed with 3.7% paraformaldehyde for 15 min, permeabilized with ice-cold methanol, then blocked with 1% bovine serum albumin (BSA). The cells were then incubated with ATR (1:100 dilution, #13934, Cell Signaling Technology) and pATR (Ser 428, 1:100 dilution, #2853, Cell Signaling Technology) primary antibodies and β-actin antibodies (1:5000 dilution, #A5316, Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. This was followed by incubation with Alexa Fluor 488 (Green) conjugated goat anti-rabbit antibody (1:1000, #A-11034, Invitrogen, La Jolla, CA, USA) and Alexa Fluor 594 (Red) goat anti-mouse antibody (1:1000, #A-11032, Invitrogen) for 1 h. Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) nuclear staining was conducted at 1:10,000 for 10 min at room temperature. Finally, the cells were imaged on a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., Sterling Heights, MI, USA) equipped with a SPOT RT™ digital camera.
3
Immunostaining Antibodies Validation
4
Western Blot Analysis of DNA Damage Signaling
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The cells were lysed in PierceTM RIPA Buffer (ThermoScientific, Waltham, MA, USA). The protein concentrations were established using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Western blotting was performed using standard procedures, and the blots were developed using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). Original images of western blot added to the Supplementary Data . Primary antibodies used were as follows: ERCC4 (#13465), p-ATM (Ser1981) (#13050), p-ATR (Ser428) (#2853), p-Chk1 (Ser345) (#2341), p-Chk2 (Thr68) (#2197), p-CDC25C (Ser216) (#9528), p-H2AX (Ser139) (#3257000) (1:1000; Cell Signaling Technology, Danvers, MA, USA); NEDD9 (ab18056, 1:1000), beta-Actin (ab49900, 1:100,000) (Abcam; Cambridge, MA, USA), Vinculin (700062) (1:1000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Vinculin and beta-Actin were used as the loading controls. The quantification was performed using NIH ImageJ Imaging Software (Rayne Rasband, National Institutes of Health, USA).
5
Multiparametric Protein Analysis Protocol
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Primary antibodies: BrDU (Millipore), E1A, 53BP1, pATMSer1981, pATRSer428, S6 ribosomal protein, pS6 ribosomal protein, p4E-BP1, Akt, pAktSer473, GAPDH, LAMP1, Nanog (all by Cell Signaling Technology); Rad51, Oct3/4 (all by Santa Cruz Biotechnology); γH2AX, pDNA-PKcsS2056 (all by Abcam); LC3 (MBL). Secondary antibodies: Alexa-fluor 488, Alexa-fluor 568 (all by Invitrogen); anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase (Sigma).
6
Molecular Mechanisms of p53-Mediated Apoptosis
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The SuperScript™ First-Strand Synthesis System and TRIzol Reagent were acquired from Invitrogen (Grand Island, USA). The plasmids of shp53 and shChk2 were obtained from Open Biosystems (Thermo Fisher Scientific, Pittsburgh, USA). Antibodies against caspase-3, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP), cleaved PARP, p53, p-p53 Ser15, p-p53 Ser20, ataxia telangiectasia mutated kinase and Rad3-related kinase (ATR), p-ATR Ser428, murine double minute 2 (MDM2), p-MDM2 Ser166, p-Chk2 Thr68, p-Chk1 Ser345, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were purchased from Cell Signaling Technology (Danvers, USA). Dexamethasone was obtained from Sigma-Aldrich (St. Louis, USA). Cycloheximide (CHX) and MG132 were acquired from Calbiochem (San Diego, USA).
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