We used antibodies against acetylated α-tubulin (Cell Signaling Technology, Danver, MA, USA, #5335), detyrosinated α-tubulin (Sigma, MAB5566), Ero1-Lα (Cell Signaling Technology, #3264), calnexin (Cell Signaling Technology, #2679), BiP (Cell Signaling Technology, #3177), IRE1α (Cell Signaling Technology, #3294), phospho-IRE1α (Cell Signaling Technology, #3398), PERK (Cell Signaling Technology, #5683), phospho-PERK (Abcam, Cambridge, MA, USA, #ab192591), ATF6 (Abcam, #ab227830), and GAPDH (Santa Cruz Biotech, TX, USA, #sc32233). Tunicamycin (Sigma, T7765) and Y-27632 (Sigma, Y0503) were purchased from Sigma-Aldrich. Blebbistatin was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).
Phospho ire1α
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-IRE1α is a lab equipment product that detects the phosphorylated form of the IRE1α protein. IRE1α is a key component of the unfolded protein response (UPR) pathway, which is involved in the cellular response to endoplasmic reticulum (ER) stress.
Automatically generated - may contain errors
Lab products found in correlation
Ire1α, by Cell Signaling Technology (4 mentions)
P erk, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Eif2α, by Cell Signaling Technology (3 mentions)
Phospho perk, by Cell Signaling Technology (2 mentions)
Phospho eif2α, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Blebbistatin, by LGC (1 mentions)
Acetylated α tubulin, by Cell Signaling Technology (1 mentions)
Calnexin, by Cell Signaling Technology (1 mentions)
Ero1 lα, by Cell Signaling Technology (1 mentions)
Phospho perk, by Abcam (1 mentions)
Y 27632, by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Caspase 3, by Merck Group (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Ire1α, by Novus Biologicals (1 mentions)
6 protocols using phospho ire1α
1
Investigating ER Stress Signaling Pathways
2
Investigating UPR Pathway Activation
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Following treatment, the cells were lysed with ice-cold Mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific, Reinach, Switzerland). The following antibodies were used: BiP (Cat. No. 3177; Cell Signalling Technology, Danvers, MA, USA), phospho-IRE1α (Cat. No. NB100-2323, Novus Biologicals, Littleton, CO, USA), IRE1α (Cat. No. 3294, Cell Signalling Technology), phospho-PERK (Cat. No. 3179S, Cell Signalling Technology), PERK (Cat. No. 3192S, Cell Signalling Technology), phospho-JNK (Cat. No. 9251, Cell Signalling Technology), JNK (Cat. No. 9252, Cell Signalling Technology), ATF6α (Cat. No. sc-166659, Santa Cruz, CA, USA), LC3 (Cat. No. L7543; Sigma-Aldrich), Caspase 3 (Cat. No. 9662, Cell Signalling Technology), PARP (Cat. No. 9542; Cell Signalling Technology) and GAPDH (Cat. No. MCA4740, BIO RAD Hercules, CA, USA). Primary antibodies were used at 1:1000 dilution for Western blotting.
Co-immunoprecipitation (Co-IP) was performed overnight at 4 °C using the IRE1α antibody (Cat. No. 3294, Cell Signalling Technology) and JNK antibody (Cat. No. 9251, Cell Signalling Technology) at 1:200 dilution. Immunocomplexes were collected with protein G sepharose beads (17-0618-01, GE Healthcare, Glattbrugg, Switzerland) for 30 min at 4 °C prior to Western blotting. Densitometry of bands was measured using ImageJ software.
Co-immunoprecipitation (Co-IP) was performed overnight at 4 °C using the IRE1α antibody (Cat. No. 3294, Cell Signalling Technology) and JNK antibody (Cat. No. 9251, Cell Signalling Technology) at 1:200 dilution. Immunocomplexes were collected with protein G sepharose beads (17-0618-01, GE Healthcare, Glattbrugg, Switzerland) for 30 min at 4 °C prior to Western blotting. Densitometry of bands was measured using ImageJ software.
3
Western Blot Analysis of UPR Markers
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Cells were washed with cold PBS and lysed with western blot lysis buffer (Beyotime, China), and the lysates were centrifuged at 13 400 g for 5 min at 4°C. Protein concentrations were quantified using a BCA Assay Kit (Beyotime, China). Proteins were separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membrane was incubated overnight at 4°C with antibodies against GAPDH (Wanleibio, China), BIP, PERK, IRE1α, phospho IRE1α, EIF2α and phospho EIF2α (all from Cell Signaling Technology, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (ZSGB-BIO, China) was incubated with the membrane at 37°C for 1 h. Proteins were detected by chemiluminescence using SuperSignal West Pico (Thermo Fisher, USA) and imaged on a ChemiDoc MP imaging system (Bio-Rad, USA).
4
Immunoblotting Analysis of UPR Signaling
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Immunoblotting was performed using 20 μg of proteins from cell lysate per sample as previously described26 (link). Primary antibodies against phospho-NF-κB p65, NF-κB p65, phospho-PERK, PERK, phospho-eIF2α, eIF2α, phospho-IRE1α, IRE1α, ATF4, and GAPDH (Cell Signaling Technology, Danvers, MA) were used in the experiments. After incubation with secondary antibody and extensive washing, blots were placed in Amersham ECL Prime Western Blotting Detection Reagent (GE Life Sciences). Blots were then imaged using the ChemiDoc MP system (Bio-Rad), and bands were quantified using ImageJ software (National Institutes of Health)26 (link). Band intensities of phospho-NF-κB p65, phospho-PERK, phospho-eIF2α, and phospho-IRE1α were normalized by band intensities of total NF-κB p65, PERK, eIF2α, and IRE1α, respectively, of the same samples on the same blot after blot stripping and re-probing. Band intensities of ATF4 were normalized by band intensities of GAPDH of the same samples on the same blot.
5
Apoptosis and ER Stress Signaling
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CBD was obtained from Sigma. CBD dissolved in absolute Ethanol (EtOH) was stored at −20 °C. CHX was purchased from Merck Milipore (Darmstadt, Germany). MG132 was purchased from Sigma. Antibodies against c-PARP, c-Cas3, -Cas8, and Cas9, Bid, Bax, p53 upregulated modulator of apoptosis, XIAP, Bip, GRP94, PERK, p-PERK, phospho-IRE1α, p-IRE1α, ATF6, CHOP, ubiquitin (Ub), and cytochrome c oxidase subunit I were purchased from Cell Signaling Technology (Danvers, MA, USA). Protein G PLUS-Agarose beads and antibodies against B-cell lymphoma-extra-large, NDUFA9, succinate dehydrogenase complex flavoprotein subunit A, RieskeFeS, and adenosine triphosphate synthase subunit α were purchased from Santa Cruz Biotechnology. β-Actin was purchased from Sigma. Antibody against p8 was purchased from abcam (Cambridge, UK). The secondary antibodies anti-mouse-IgG-horseradish peroxidase (HRP) and anti-rabbit-IgG-HRP were purchased from Cell Signaling Technology.
6
Immunoblotting Analysis of Apoptosis and UPR Signaling
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Cells were harvested after treatment, washed 3 times with PBS and lysed in CHAPS lysis buffer (Cell Signaling Technology). The concentration of protein was measured using the BCA protein assay kit, and loading buffer was added to the samples. The samples were subjected to 12% SDS-PAGE; then proteins were transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk at room temperature for 1 h, PVDF membranes were incubated with primary antibodies overnight at 4°C. The following antibodies against these proteins were used: Caspase-3, PARP, Mcl-1, Bcl-xl, Bcl-2, Bax, Smac/DIABLO, Cytochrome C, ATF-4, phospho-IRE1α, IRE1α, phosphor-eIF2α, eIF2α, phosphor-Akt, Akt, phosphor-PI3K, PI3K, mTOR, and GAPDH, which were all purchased from Cell Signaling Technologies (Danvers, USA). Next, the membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature.
Finally, the proteins were detected by enhanced chemiluminescence, and densitometric analysis was performed using ImageJ software. The cytosolic fraction of the cells was purified as described earlier [25] .
Finally, the proteins were detected by enhanced chemiluminescence, and densitometric analysis was performed using ImageJ software. The cytosolic fraction of the cells was purified as described earlier [25] .
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