Immunoblot analysis was performed according to the standard procedures using the following antibodies and dilutions: Anti‐apoptosis signalling regulating kinase1 (ASK1; Sc‐7931), 1:500; anti‐ p‐ASK1 (Thr 845, Sc‐109911), 1:1000; anti‐ JNK (Sc‐474), 1:1000; anti‐ p‐JNK (SC‐6254), 1:1000; anti‐ p38 (Sc‐535), 1:1000; anti‐ p‐p38 (SC‐7973), 1:1000; anti‐ Noxa (SC‐2697), 1:1000; anti‐Mcl‐1 (SC‐20679), 1:500; anti‐ Actin (Sc‐1615), 1:5000; anti‐ Tom20 (Sc‐11415), 1:100; anti‐ Bap31 (Sc‐17764), 1:500; anti‐ IRE1α (Sc‐20790), 1:500; anti‐ protein kinase RNA‐like endoplasmic reticulum kinase (PERK; SC‐9477), 1:1000; anti‐ p‐ERK (SC‐32577), 1:1000; anti‐ calpain 1 (Sc‐7530), 1:1000; anti‐ caspase‐4 (Sc‐1229), 1:1000; anti‐ ATF4 (SC‐200), 1:1000; anti‐ XIAP (SC‐11426), 1:1000; anti‐ IκB‐α (sc‐203), 1:500; anti‐ p‐IκB‐α (sc‐8404), 1:500 (from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti‐ p‐IRE1α (PA1‐16927; Thermo Scientific, FL 33407, USA), 1:1000; anti‐ CHOP (#28950, 1:1000; anti‐ caspase 3 (#7190), 1:1000; anti‐ caspase 9 (#9501), 1:1000; anti‐ PARP (#9542), 1:500; anti‐ Erk1/2 (#9102), 1:1000; anti‐ p‐Erk1/2 (#9101), 1:1000 (from Cell Signalling Technology Inc., Danvers, MA, USA); anti‐ p‐IRE1 (ab48187), 1:1000; anti‐ p‐ATF‐4 (ab28830), 1:1000; anti‐GRP78 (ab108613), 1:500 (from Abcam, Massachusetts, USA).
Anti p ire1α
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti–p-IRE1α is a primary antibody used to detect the phosphorylated form of the IRE1α (Inositol-requiring enzyme 1α) protein. IRE1α is a key component of the unfolded protein response (UPR) pathway, which is activated in response to endoplasmic reticulum (ER) stress. The Anti–p-IRE1α antibody can be used in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the activation and regulation of the IRE1α pathway.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Anti ire1α, by Cell Signaling Technology (3 mentions)
Anti atf6, by Abcam (2 mentions)
Anti hsp90α, by Cell Signaling Technology (2 mentions)
Anti hsp90β, by Cell Signaling Technology (2 mentions)
Bicinchoninic acid assay, by Beyotime (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti grp78, by Cell Signaling Technology (2 mentions)
Anti caspase 12 ab62484, by Abcam (2 mentions)
Anti grp78, by Abcam (2 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Anti p iκb α, by Santa Cruz Biotechnology (1 mentions)
Anti ubiquitin p4d1, by Santa Cruz Biotechnology (1 mentions)
Flag m2 affinity gel, by Merck Group (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti chop, by Cell Signaling Technology (1 mentions)
Anti vdac, by Cell Signaling Technology (1 mentions)
Anti bip, by Cell Signaling Technology (1 mentions)
Z lehd fmk, by R&D Systems (1 mentions)
5 protocols using anti p ire1α
1
Immunoblot Analysis of Stress Signaling
2
Western Blot Analysis of ER Stress Markers
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The protocol of Western blotting was similar with our previous study (65 (link)). The brain tissue or cell sample was sonicated in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitors. Samples were centrifuged at 12,000g for 20 min at 4°C, and the protein concentrations were examined by the bicinchoninic acid assay (Beyotime Biotechnology, Haimen, China). All the protein samples were heated for 5 min at 95°C in loading buffer. The deactivated protein samples were separated by SDS–polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The transferred membranes were incubated with primary antibodies and secondary antibodies. Anti-ATF6 (#ab37149; 1:500), anti–caspase 12 (ab62484; 1:1000), and anti-GRP78 (#ab21685; 1:1000) were purchased from Abcam (Cambridge, MA, USA). Anti-IRE1α (#3294; 1:500), anti-GRP78 (#3183; 1:1000), anti-HSP90α (#8165; 1:1000), and anti-HSP90β (#7411; 1:1000) were obtained from Cell Signaling Technology (San Francisco, CA, USA). Anti–p-IRE1α (#PA1-16927; 1:500) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti–β-actin (#sc-47778; 1:2000) was obtained from Santa Cruz Biotechnology (Waltham, MA, USA).
3
Ubiquitylated Protein Enrichment and Analysis
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For the proteomic screen, ubiquitylated proteins were pulled-down with TUBEs 1 (LifeSensors, Malvern, PA, USA) and UbiQapture (Enzo Life Sciences, France) according to the manufacturer's instructions.
FLAG-tagged proteins were immunoprecipitated with FLAG M2 affinity gel (Sigma Aldrich, St Louis, MO, USA) and eluted two times with lysis buffer+0.05 mg of FLAG peptide at 4 °C. IκB was immunoprecipitated using anti-IκB conjugated to agarose beads (CST, Danvers, MA, USA). Tandem affinity purification of ubiquitylated proteins in denaturing conditions (8 M urea) were performed as described in Meierhofer et al.43 (link)The following antibodies were used: anti-ubiquitin (P4D1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IκB (CST), anti-GAPDH (G9545; Sigma Aldrich), streptavidin-HRP (Thermo Fisher Scientific, Waltham, MA, USA), anti-C3a (BD), and anti-p-IRE1α (Thermo Fisher Scientific).
FLAG-tagged proteins were immunoprecipitated with FLAG M2 affinity gel (Sigma Aldrich, St Louis, MO, USA) and eluted two times with lysis buffer+0.05 mg of FLAG peptide at 4 °C. IκB was immunoprecipitated using anti-IκB conjugated to agarose beads (CST, Danvers, MA, USA). Tandem affinity purification of ubiquitylated proteins in denaturing conditions (8 M urea) were performed as described in Meierhofer et al.43 (link)The following antibodies were used: anti-ubiquitin (P4D1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IκB (CST), anti-GAPDH (G9545; Sigma Aldrich), streptavidin-HRP (Thermo Fisher Scientific, Waltham, MA, USA), anti-C3a (BD), and anti-p-IRE1α (Thermo Fisher Scientific).
4
Western Blot Analysis of ER Stress Markers
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The protocol of Western blotting was similar with our previous study (65 (link)). The brain tissue or cell sample was sonicated in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitors. Samples were centrifuged at 12,000g for 20 min at 4°C, and the protein concentrations were examined by the bicinchoninic acid assay (Beyotime Biotechnology, Haimen, China). All the protein samples were heated for 5 min at 95°C in loading buffer. The deactivated protein samples were separated by SDS–polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The transferred membranes were incubated with primary antibodies and secondary antibodies. Anti-ATF6 (#ab37149; 1:500), anti–caspase 12 (ab62484; 1:1000), and anti-GRP78 (#ab21685; 1:1000) were purchased from Abcam (Cambridge, MA, USA). Anti-IRE1α (#3294; 1:500), anti-GRP78 (#3183; 1:1000), anti-HSP90α (#8165; 1:1000), and anti-HSP90β (#7411; 1:1000) were obtained from Cell Signaling Technology (San Francisco, CA, USA). Anti–p-IRE1α (#PA1-16927; 1:500) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti–β-actin (#sc-47778; 1:2000) was obtained from Santa Cruz Biotechnology (Waltham, MA, USA).
5
Exploring Apoptosis Pathways in Cells
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Synthetic N-hexanoyl-DL-homoserine lactone (C6-HSL), N-decanoyl-DL-homoserine lactone (C10-HSL), N-(3-hydroxy dodecanoyl)-DL-homoserine lactone (OH-dDHL), tunicamycin, thapsigargin, hydrogen peroxide 30% (wt/vol) (H2O2), N-Acetyl-l-cysteine (NAC), and staurosporine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies with anti-caspase-3, anti-caspase-8, anti-cytochrome c, anti-PARP, anti-β-actin, anti-caspase-9, anti-VDAC, anti-CHOP, anti- caspase-12, anti-p-eIF2α, anti-IRE1α, and anti-BIP were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p-IRE1α was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-ATF 6α, anti-COX-2, and methyl-β-cyclodextrin (MβCD) were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Abcam (Cambridge, MA, USA). The pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVE-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) were purchased from R&D Systems (Minneapolis, MN, USA). The paraoxonase 2 (PON2) inhibitor ((1,2,4)trizolo(4,3–a)quinolones; TQ416) was purchased from Maybridge (Cornwall, UK).
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