AF568-Annexin V, MitoTracker Deep Red, Protein A/G magnetic beads and AF680 conjugated goat anti-rabbit IgG (A21109) were purchased from Invitrogen (Burlington, ON, Canada). FluidMAG-DXS beads were from Chemicell (Berlin, Germany). Mortalin (sc-133137) and β-actin (sc-1616-R) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PARP antibody (9542) was from Cell Signaling (Denvers, MA, USA). FITC-conjugated goat anti-rabbit IgG (L43001) was from Caltag (Burlingame, CA, USA). DLST antibody (HPA003010), MKT-077 and paraformaldehyde (PFA) were from Sigma-Aldrich (St. Louis, MO, USA). Vir S antibody (ab22743) was purchased from Abcam (ON, Canada). Ultra-small goat anti-rabbit IgG (25101) was from Electron Microscopy Sciences (Hatfield, PA, USA). Staurosporine was purchased from Calbiochem (San Diego, CA, USA). Recombinant Cpn60.2 protein and Cpn60.2 antibody (kindly provided by Dr Richard W. Stokes, University of British Columbia, Canada) were described earlier (Hickey et al., 2010 (link)) and Dos R antibody was kindly provided by Dr Yossef Av-Gay, University of British Columbia, Canada.
Parp antibody 9542
Manufactured by Cell Signaling Technology
Sourced in United States
The PARP antibody (9542) is a research-use antibody developed by Cell Signaling Technology. It is designed to detect the presence of Poly(ADP-ribose) Polymerase (PARP) protein, which is involved in various cellular processes. The antibody can be used in immunoassays and other applications to study the expression and distribution of PARP in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde pfa, by Merck Group (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Mkt 077, by Merck Group (1 mentions)
β actin sc 1616 r, by Santa Cruz Biotechnology (1 mentions)
Mortalin sc 133137, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3 antibody 9661, by Cell Signaling Technology (1 mentions)
Arsenic trioxide, by Merck Group (1 mentions)
Sc966, by Fortis Life Sciences (1 mentions)
Glut1 antibody, by Thermo Fisher Scientific (1 mentions)
C myc antibody sc 40, by Santa Cruz Biotechnology (1 mentions)
Specific antibodies, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 asp175 5a1e, by Cell Signaling Technology (1 mentions)
Mdm2 d1v2z, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Nacalai Tesque (1 mentions)
5 protocols using parp antibody 9542
1
Molecular Probes for Cell Death Analysis
2
Protein Expression Analysis by SDS-PAGE and Western Blot
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SDS-PAGE Electrophoresis and Western Blotting were performed. Briefly, cells were washed in PBS and lysed for about 30 min in lysis buffer containing 15 mM Tris/HCl pH 7.5, 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton ×100 and Protease Inhibitor Cocktail (100×, Sigma-Aldrich, St. Louis, MO, USA). Cell lysates (30 µg per lane) were separated using Bolt Bis-Tris gel 4–12% (Thermo Fisher Scientific, Cambridge, MA, USA) and transferred on Nitrocellulose membranes (GE Healthcare, Milan, Italy); the membrane was incubated in blocking solution (5% BSA, 20 mM Tris, 140 mM NaCl, 0.1% Tween-20) and probed overnight with the specific antibodies.
The antibodies against the following proteins were used: E-Cadherin (24E10) Rabbit mAb 3195, Vimentin (D21H3) XP® Rabbit mAb 5741, Snail (C15D3) Rabbit mAb #3879, p38 MAPK Antibody #9212 and Phospho-p38 MAPK (Thr180/Tyr182) Antibody #9211, PARP Antibody 9542, and Apoptosis Antibody Sampler Kit #9915 all from Cell Signaling (Cell Signaling, Beverly, MA, USA); α Tubulin TU-02 (sc-8035) from Santa-Cruz Biotechnology (Santa Cruz Biotechnology, Inc. Dallas, TX, USA). The membranes were incubated with secondary antibody Dylight 488 (Thermo Fisher Scientific, Cambridge, MA, USA) and signal was detected by Chemidoc (Biorad, Milan, Italy).
The antibodies against the following proteins were used: E-Cadherin (24E10) Rabbit mAb 3195, Vimentin (D21H3) XP® Rabbit mAb 5741, Snail (C15D3) Rabbit mAb #3879, p38 MAPK Antibody #9212 and Phospho-p38 MAPK (Thr180/Tyr182) Antibody #9211, PARP Antibody 9542, and Apoptosis Antibody Sampler Kit #9915 all from Cell Signaling (Cell Signaling, Beverly, MA, USA); α Tubulin TU-02 (sc-8035) from Santa-Cruz Biotechnology (Santa Cruz Biotechnology, Inc. Dallas, TX, USA). The membranes were incubated with secondary antibody Dylight 488 (Thermo Fisher Scientific, Cambridge, MA, USA) and signal was detected by Chemidoc (Biorad, Milan, Italy).
3
Immunoblotting and Immunofluorescence Assays
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PML antibodies were purchased from Santa Cruz (sc-966) for immunofluorescent staining, or from Bethyl (A301–167A) for immunoblotting. c-Myc antibody (sc-40) and Sox2 antibody (sc-17320) were purchased from Santa Cruz. PARP antibody (9542) and cleaved Caspase-3 antibody (9661S) were from Cell Signaling. Glut1 antibody was from Thermo Scientific (PA1–37782). Arsenic trioxide was purchased from Sigma (202673–5G) and the stock solution was prepared at 100 mM as instructed by manufacturer.
4
Multimodal Regulation of p53 Signaling
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The Western blotting procedure and antibodies for the target proteins were described previously [22 (link)]. In vitro assays for ubiquitination by MDM2B-MDM4 were performed as described previously with minor modifications [15 (link)]. Briefly, the reactions were carried out at 30 °C for 1 h in a volume of 20 μL reaction in the presence of different concentrations of compounds or vehicle solvent DMSO, followed by a WB of p53 with DO-1, MDM2 with rabbit anti-MDM2 (MDM2 (D1V2Z) (#86934, Cell Signaling Technology, Danvers, MA 01923, USA), or MDM4 with a rabbit anti-MDM4 antibody (Proteintech, Rosemont, IL 60018, USA, Cat no: 17914-1-AP). The apoptotic response to compounds was measured by Western blotting using specific antibodies from Cell Signaling Technology, Danvers, MA 01923, USA) for activated caspase 3 (Cleaved Caspase-3 (Asp175) (5A1E) (#9664) and PARP (PARP Antibody #9542).
5
Western Blotting Techniques and Antibodies
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Cell were lysed with TNE lysis buffer containing, 0.1% SDS, 50 mM Trs-HCl, 120 mM NaCl , 5 mM EDTA, 1% NP-40, and 10-20% Protease inhibitor (Nakalai Tesque, Kyoto, Japan or Wako). Western blotting was basically performed as previously reported 58 . TBS buffer containing 5% skim milk or BSA fraction V and 0.1% Tween-20 was used for blocking. For some blots, Can Get Signal® Immunoreaction Enhancer Solution (TOYOBO, Osaka, Japan) was used for enhancement of detection. We used an antibody against CNOT3 which is commercially available (H00004849-M01, Abnova, Taipei, Taiwan). Antibodies against CNOT1, CNOT2, CNOT6L, CNOT7 were described previously 10 . Rabbit CNOT9 antibody was obtained as described previously 59 .
We purchased RB (554136) antibody from BD Biosciences and PARP antibody (#9542) from Cell Signaling Technology, MA, USA. Goat Lamin B antibody (sc-6217), mouse p53 (sc-126) and -actin (sc-69879) antibodies were from Santa Cruz, TX, USA. p21 antibodies were from BD biosciences (556430) and Santa Cruz (sc-6246), and p27 antibodies were from Santa Cruz (sc-528) and abcam, Cambridge, England (ab32034).
-tubulin (T9026) was purchased from Sigma-Aldrich, MO, USA. Quantification of the bands was performed using Image J in a vertical way. When it was impossible to measure in a vertical way because of the continuous band, it was performed in a horizontal way.
We purchased RB (554136) antibody from BD Biosciences and PARP antibody (#9542) from Cell Signaling Technology, MA, USA. Goat Lamin B antibody (sc-6217), mouse p53 (sc-126) and -actin (sc-69879) antibodies were from Santa Cruz, TX, USA. p21 antibodies were from BD biosciences (556430) and Santa Cruz (sc-6246), and p27 antibodies were from Santa Cruz (sc-528) and abcam, Cambridge, England (ab32034).
-tubulin (T9026) was purchased from Sigma-Aldrich, MO, USA. Quantification of the bands was performed using Image J in a vertical way. When it was impossible to measure in a vertical way because of the continuous band, it was performed in a horizontal way.
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