WB analysis was performed as described previously.[35 (link)] The antibodies used in the experiments were rabbit anti-PAX5 (#12709, 1:1000), mouse p53 (1C12) (#2524, 1:1000), rabbit poly adenosine disphosphate-ribose polymerase (PARP) (46D11) (#9532, 1:1000), rabbit anti-caspase-3 (#9661, 1:1000), rabbit anti-caspase-7 (#8438, 1:1000), rabbit anti-caspase-8 (#9496, 1:1000), rabbit anti-caspase-9 (#7237, 1:1000), PARP (#5625, 1:1000) (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-p53 antibody (ab131442, 1:1000), rabbit anti-pro Caspase-3 antibody (ab32150, 1:1000), rabbit anti-pro Caspase-7 antibody (ab32067, 1:1000), rabbit anti-Pro Caspase-8 antibody (ab108333, 1:1000), rabbit anti-pro Caspase-9 (phospho T125) (ab138412, 1:500), and rabbit anti-PAX5 antibody (ab109443, 1:1000) (Abcam, Cambridge, UK). Rabbit anti-actin was used as a control (Cell Signaling Technology).
Parp 46d11
Manufactured by Cell Signaling Technology
Sourced in United States
PARP (46D11) is a primary antibody that recognizes the Poly(ADP-ribose) Polymerase (PARP) protein. PARP is an enzyme involved in various cellular processes, including DNA repair and programmed cell death. The 46D11 clone of this antibody can be used to detect and study the PARP protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti actin, by Cell Signaling Technology (1 mentions)
Ab109443, by Abcam (1 mentions)
Ab32150, by Abcam (1 mentions)
Ab108333, by Abcam (1 mentions)
Ab131442, by Abcam (1 mentions)
Rabbit anti caspase 9, by Cell Signaling Technology (1 mentions)
Rabbit anti caspase 7, by Cell Signaling Technology (1 mentions)
Rabbit anti pax5, by Cell Signaling Technology (1 mentions)
Rabbit anti caspase 8, by Cell Signaling Technology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Proteintech (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Gfp sc 8334, by Santa Cruz Biotechnology (1 mentions)
15 protocols using parp 46d11
1
Apoptotic Signaling Pathway Evaluation
2
Validation of Western Blot and qPCR Protocols
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The procedures for Western blot and RT-qPCR were well established as described previously.30 (link),31 (link) Antibodies used for western blot are poly (ADP-ribose) polymerase (PARP) (46D11) (#9532; Cell Signaling Technology, Danvers, MA) and β-actin (sc-47778, C4; Santa Cruz Biotechnology, Santa Cruz, CA). The primers used in qPCR are Pdk4 (forward: 5′-TCCCCGCTGTCCATGAAG-3’; reverse: 5′-CGTTCTTTCACAGGCATTTTCTG-3′), Tnf (forward: 5′-CAGCCGATGGGTTGTACCTT-3’; reverse: 5′-GGCAGCCTTGTCCCTTGA-3′), baculoviral IAP repeat-containing 3 (Birc3) (forward: 5′-TGGGTCAGTCTGCTTCGAGAT-3’; reverse: 5′-AATACGGGCTGCGTGTGTCT-3′), interferon gamma (Ifnγ) (forward: 5′-TTGGCTTTGCAGCTCTTCCT-3’; reverse: 5′-TGACTGTGCCGTGGCAGTA-3′), E-selectin (forward: 5′-CTTGCATGGCTCAGCTCAAC-3’; reverse: 5′-GGGACTTCCTGGGTCCACTT-3′), growth arrest and DNA-damage-inducible beta (Gadd45β) (forward: 5′-CGTTCTGCTGCGACAATGAC-3’; reverse: 5′-GCGCCAGCCTCTGCAT-3′), coiled-coil domain containing 103 (Ccdc103) (forward: 5′-AGCCATGCAGAGCGAGAGA-3’; reverse: 5′-TGCTCATGGCTTGCAACTTC-3′) and Actb (forward: 5′-CGATGCCCTGAGGCTCTTT-3’; reverse: 5′-TGGATGCCACAGGATTCCA-3′). For mouse study, both individual and pooled protein and RNA samples were compared to validate the difference between groups.
3
Immunoblotting Protein Detection Protocol
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Samples were resolved by SDS-PAGE (10–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated. Primary antibodies were used to detect Bcl-xL (54H6, 1:1,000 dilution, Cell Signaling; 2H12, 1:500 dilution, Invitrogen), COX IV (3E11, 1:20,000 dilution, Cell Signaling), Drp1 (D6C7, 1:1,000 dilution, Cell Signaling), Flag (66008-3-Ig, 1:1,000 dilution, Proteintech), GAPDH (sc-365062, 1:1,000 dilution, Santa Cruz biotechnology), GFP (sc-8334, 1:1,000 dilution, Santa Cruz biotechnology), HA (51064-2-AP, 1:1,000 dilution, Proteintech), Mff (17090-1-AP, 1:2,000 dilution, Proteintech), PARP (46D11; 1:1,000 dilution, Cell Signaling), RFP (3F5, 1:1,000 dilution, ChromoTek), SENP3 (D20A10, 1:10,000 dilution, Cell Signaling), and β-actin (A2228, 1:20,000 dilution, Sigma). Immune complexes were detected either using HRP-conjugated secondary antibodies (Sigma) or an HRP-conjugated VeriBlot secondary antibody (ab131366, Abcam; for immunoblotting involving IP samples) followed by enhanced chemiluminescence (GE Healthcare Amersham), or using fluorescent secondary antibodies (Thermo Fisher Scientific) by a LI-COR imaging system. Each immunoblot presented is representative of at least three independent experiments carried out using different cell populations.
4
Western Blot Analysis of PARP Protein
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Protein extraction was conducted via TRIzol according to the manufacturer's recommendations. Polyacrylamide gels were prepared by combining 40% acryl to 2% Bis, 1 M Tris pH 8.7, 1 M Tris pH 6.9, 20% SDS and water (All Sigma-Aldrich, St. Louis, MO, USA). TEMED (Sigma-Aldrich, St. Louis, MO, USA) and 10% Ammonium Persulfate (Serva, Heidelberg Baden-Württemberg, Germany) were added immediately prior to gel casting. Gels were run at 150 V for 90 min and blotted on a Nitrocellulose membrane (Thermo Fisher Scientific, Vilnius, Vilniaus County, Lithuania) at 150 V for 60 min. Membranes were blocked in 5% skimmed milk Tris Buffer Saline (TBS) solution with 1% Tween (Serva, Heidelberg Baden-Württemberg, Germany) blocking solution for 1 h. Membrane was then soaked overnight in primary antibody PARP (46D11) (mAb #9532, Cell Signaling Technologies, USA) in blocking solution on a rocker. Membranes were washed with TBS Tween and subject to goat anti-rabbit secondary antibodies (Thermo Fisher Scientific, Vilnius, Vilniaus County, Lithuania) in blocking solution for 2 h, washed with TBS Tween, followed by BCPI/NBT phosphatase substrate (Serva, Heidelberg Baden-Württemberg, Germany) detection.
5
Comprehensive Antibody Panel for Protein Analysis
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General RSK1 (C-21; 1:4000), pRSK1/2 (T359, S381; 1:2000), pcMyc (T58, S62; 1:1000) and GAPDH (FL-335; 1:1000) antibodies (Abs) were obtained from Santa Cruz Biotechnology (CA, USA). Anti-CyclinD1 (92G2, 1:1000) and PARP (46D11, 1:1000) Abs were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-pERK1/2 (1:20000) and gERK1/2 (1:20000) Abs were obtained from Sigma (Rehovot, Israel). Anti-Sprouty2 (aminoterminal, 1:1000) Ab was obtained from Abcam (Cambridge, UK). Secondary fluorescent Ab conjugates were obtained from Jackson ImmunoResearch (West Grove, PA). Secondary Abs conjugated to horseradish peroxidase (HRP) were obtained from Nichirei Biosciences (Japan).
6
Synthesis and Evaluation of Compounds 1-3
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Compounds 1 , 2 , and 3 were synthesized according to the procedure developed by our group [12 (link)]. They were dissolved in dimethyl sulfoxide (DMSO), the concentration of which never exceeded 0.1% (v/v); 50 mM of stock solution was stored at −20 °C. The Cell counting Kit-8 (CCK-8), Hoechst 33258, JC-1, and the ROS assay kit were purchased from Beyotime Institute of Biotechnology Company (Shanghai, China). The annexin V-FITC and propidium iodide (PI) kit was obtained from BD Pharmingen (BD, San Diego, CA, USA). The primary antibodies used are as follows: CDK4 (D9G3E, Cell Signaling Technology, Danvers, MA, USA), Cyclin D1 (E3P5S, Cell Signaling Technology), Caspase-3 and cleaved Caspase-3 (D3R6Y, Cell Signaling Technology), PARP (46D11, Cell Signaling Technology), Cytochrome c (AF0146, Affinity, Changzhou, China), Caspase-9 (AF6348, Affinity), cleaved Caspase-9 (D8I9E, Cell Signaling Technology), BAX (D2E11, Cell Signaling Technology), Bcl-2 (D17C4, Cell Signaling Technology), Bad (D24A9, Cell Signaling Technology), and β-actin (AF0003, Beyotime Biotechnology, Shanghai, China). All other chemicals used are commercially available and reagent grade.
7
EGFR Signaling Pathway Inhibition
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The following materials were commercially obtained: osimertinib (Chemscene, Monmouth Junction, NJ, USA), gefitinib (Wako Pure Chemical Industries, Osaka, Japan), erlotinib (Wako Pure Chemical Industries), and DMSO (Wako Pure Chemical Industries). Antibodies were obtained from the following sources: EGFR [EP38Y] (Abcam, Cambridge, UK), PARP [46D11] (Cell Signaling Technology, Beverly, MA, USA), EphB4 [D1C7N] (Cell Signaling Technology), β-actin [AC-15] (Sigma-Aldrich, Burlington, MO, USA), and Ki-67 [MIB1] (DAKO, Santa Clara, CA, USA).
8
Validation of Western Blot and qPCR Protocols
9
Autophagy Modulation by 3-MA and CQ
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3-Methyladenine (3-MA) and chloroquine (CQ) (Sigmae-Aldrich, St. Louis, MO, USA) were prepared and diluted with DMEM. Cyto-ID Green dye was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). 3- (4, 5-dimethylthiazol-2-y1) -2, 5-diphenyltetrazolium bromide (MTT) was purchased from Biotech Well (Shanghai, China). Anti-LC3B (D11), anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-AKT (Ser473), anti-AKT, and PARP (46D11) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit immunoglobulinG (IgG) were obtained from Biotech Well (Shanghai, China).
10
Protein Extraction and Western Blot Analysis
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For protein extraction, 1×106 cells were lysed in 200 μl RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS) containing Complete Protease Inhibitor Cocktail (Roche). Cell lysates were centrifuged at 14,000 × g for 30 min at 4°C, then supernatant was harvested. Proteins were quantified by use of the Bio-Rad DC Protein Assay kit, separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blotted with antibodies for UBE2C (1∶1000, H00011065-M01, Abnova), Bcl-2 (1∶1000, 1017-1, Epitomics), and poly (ADP-ribose) polymerase (PARP; 46D11), Bcl-XL (2762), caspase-3 (8G10), caspase-8 (D35G2) and caspase-9 (all 1∶1000, C9, all Cell Signaling). Incubation with anti-β-actin (1∶5000, MAB1501, Millipore) was a loading control.
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