Western blots were performed to detect protein levels in the ipsilateral cerebral cortex and primary microglia cells. The ipsilateral cerebral cortex tissues were homogenized and lysed with RIPA buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Abcam) and treated cells were lysed using a Mammalian Cell Lysis kit (Sigma). The extracted proteins were then separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide and electrically transferred to PVDF membranes (Millipore). The membranes were then blocked with TBST with 5% non-fat dry milk for 1 h at room temperature. The western blots were probed with primary antibodies recognizing the following proteins and further incubated with corresponding secondary antibody (1:10000; Invitrogen): PTP1B (1:1000; Abcam); p-PERK (Thr982, 1:1000; Invitrogen); PERK (1:1000; Invitrogen); p-eIF2α (Ser51, 1:1000; Invitrogen); eIF2α (1:1000; Invitrogen); p-IRE1 (Ser724, 1:1000; Abcam); IRE1 (1:2000; Abcam); ATF6 (1:2000; Abcam); LC3-I/II (1:1000; Cell Signaling); beclin-1 (1:1000; Cell Signaling); β-actin (1:5000; Abcam); and GAPDH (1:5000; Abcam). The levels of protein expression were analyzed using Image J software (version 1.49) normalized to β-actin and GAPDH. Phosphorylated protein levels were evaluated compared to total protein levels.
P ire1
Manufactured by Abcam
Sourced in United Kingdom, United States
P-IRE1 is a laboratory equipment product. It serves a core function related to its intended use, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Cell Signaling Technology (3 mentions)
β actin, by Proteintech (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
P perk, by Cell Signaling Technology (2 mentions)
Caspase 12, by Abcam (2 mentions)
Grp78, by Cell Signaling Technology (2 mentions)
Xbp1s, by Abcam (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti grp78, by Abcam (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Ptp1b, by Abcam (1 mentions)
Eif2α, by Thermo Fisher Scientific (1 mentions)
P eif2α, by Thermo Fisher Scientific (1 mentions)
10 protocols using p ire1
1
Evaluating UPR Protein Expression
2
Immunoblotting Analysis of Inflammatory Markers
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Western blot analysis was conducted according to previously described procedures [61 (link)] with the following antibodies: IL-1β (Proteintech, Beijing, China); IL-18 (Abcam, Cambridge, UK); NLPR3 (Abcam, Cambridge, UK); Caspase-1 (Abcam, Cambridge, UK); NF-κB p65 (Abcam, Cambridge, UK); cytochrome C (Abcam, Cambridge, UK); GRP78 (Proteintech, Beijing, China); p-IRE1 (Abcam, Cambridge, UK); p-eif2α (Abways, Shanghai, China); ATF6 (Abcam, Cambridge, UK); P62 (Abcam, Cambridge, UK); LC3 (Abcam, Cambridge, UK); and, β-actin (Proteintech, Beijing, China). Binding of antibodies was detected using HRP-labeled goat anti-rabbit (ZHHC, Shaanxi, China) or goat anti-mouse (ZHHC, Shaanxi, China) immuno-globulin. The bands were imaged subsequently using a chemiluminescent gel imaging system (Bio-Rad, Hercules, CA, USA).
3
Apoptosis Pathway Protein Assay
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GRP78, CHOP, PERK, p-PERK, eIF2α, BCL-2, Bax, cytochrome c, caspase-3, p-LKB1, LKB1, p-AMPK, AMPK, PARP and p- eIF2α antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and IRE1, p-IRE1, XBP1s,Fas, FasL, FADD, caspase-8 and caspase-12 antibodies were purchased from Abcam (Cambridge, UK) while β-actin, PGC1-α and ATF6 antibodies were purchased from Santa Cruz (CA, USA). Unless otherwise noted, all chemical reagents were purchased from Sigma (St. Louis, MO, U.S.A).
4
ER Stress Pathway Activation Assay
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Antibodies against eIF-2a and CHOP were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Antibodies against p-eIF2α, Bcl-2, Bax, Caspase12, and p-IRE1 were purchased from Abcam Technology (Cambridge, UK). Antibodies against PERK, p-PERK, Cleaved-Caspase3, Caspase8, and Caspase9 were purchased from Cell Signaling Technology (Denver, USA). Antibodies against ATF6 were purchased from Wanlei Biology Technology (Shenyang, China). Cigarettes were acquired from Marlboro, USA. UA was bought from Wanxianghengyuan Biotechnology (Tianjin, China), and dissolved in phosphate-buffered saline (PBS) with 1% Tween80 before use. The Bicinchoninic acid (BCA) protein assay kit was acquired from Pierce (Thermo scientific, Rockford, USA), and the super enhanced chemiluminescence kit was purchased from Applygen Technologies Inc. (Beijing, China).
5
Bile Acid-Induced ER Stress Signaling
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HET1A cells were treated with bile acids for 6 h. For PP2 pre-treatment studies PP2 (10 uM) was added 24 h prior to co-treatment with bile acids for 6 h. For salubrinal and GSK2606414 pre-treatment studies, these compounds were added 1 h prior to co-treatment with bile acids. Cells were lysed with NP40 buffer and protein quantified using a PierceTM BCA kit (Thermo Fisher Scientific Waltham, MA USA). Equal concentrations of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% Marvel, primary antibodies; pPERK/Total PERK/total IRE-1/pSrc/total src/pEFI2a/total EIF2α/tubulin/actin (Cell signalling Technologies Inc. Danvers, MA, USA) or pIRE-1 (Abcam, Cambridge, United Kingdom) were incubated overnight in 5% BSA/TBST. HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc. Dallas, Texas) were incubated for 1 h and detected using PierceTM ECL substrate (Thermo Fisher Scientific, Waltham, MA USA).
6
Endoplasmic Reticulum Stress Signaling Pathway Assay
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Total protein was collected from cells using a Total Protein Extraction Kit (Sangon Biotech, Shanghai, China). Equal amounts of protein per sample were separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk for 1 hour at 37°C and then incubated at 4°C overnight with primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 hour. Primary antibodies against PDIA3, ATF-6α, p-PERK (Santa Cruz, CA), GRP78, CHOP, PERK, IRE1, SREBP1, FAS, ACC1 (Cell Signaling Technology, Danvers, MA) and p-IRE1 (Abcam, UK) were selected for this study. GAPDH (Cell Signaling Technology) was used as a control. The secondary antibodies were purchased from Santa Cruz Biotechnology. The immunoreactive bands were detected with ECL reagents and exposed to x-ray film for visualization. The density of each protein band was quantified by normalization to GAPDH.
7
Western Blot Analysis of ER Stress Proteins
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Western blot analysis was conducted according to previously described procedures [54 (link)] with antibodies against the following proteins: GRP78 (Abcam, Cambridge, UK), P-IRE1 (Abcam), p-eIF2α (Abways Techology, Shanghai, China), PERK (Santa, Shanghai, China), Nrf2 (Proteintech, Wuhan, China), ATF4 (Santa), ATF6 (Abcam), β-actin (Proteintech), HRP-labeled goat anti-rabbit (ZHHC, Xi’an, China) or goat anti-mouse (ZHHC) immunoglobulin, Caspase3 (Abways Techology), CHOP (Abmart, Shanghai, China), or p65 (Abcam). Grayscale analysis was performed using Image J software (Version 1.8.0; Bethesda, Rockville, MD, USA).
8
Hippocampal Protein Analysis Protocol
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The ipsilateral hippocampus was isolated and kept at -80℃ immediately until use. The RIPA lysis buffer was employed to homogenize the and further span at 14,000 rpm at 4℃ for 30 minutes. We collected the supernatant and then employed the BCA Protein Assay Kit to quantify the proteins. The proteins were then fractionated on a 10% SDS-PAGE gel. After that, the fractionated proteins were blotted onto PVDF membranes. Then, 5% BSA was employed to block the membranes. After that, the membranes were inoculated with the primary antibodies consisting of; anti-GRP78 (1:500, Abcam, UK), anti-LC3B (1:500, Cell Signaling Technology, USA), p62 (1:500, Cell Signaling Technology, USA), IRE-1 (1:500, Abcam, UK), p-IRE-1 (1:500, Abcam, UK), JNK (1:500, Abcam, UK), p-JNK (1:500, Cell Signaling Technology, USA), beclin1 (1:500, Cell Signaling Technology, USA) overnight at 4°C. Thereafter, the membranes were inoculated with speci ed secondary anti-bodies for 2 hours at room temperature. Immunolabeling was done with the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scienti c, USA). The GE Amersham Imager 600 was employed to image the immunoblots and the Image J software utilized to analyze the protein bands. β-actin served as the normalization standard.
9
Western Blot Analysis of EMT and Cell Cycle Markers
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The breast and pancreatic cancer cells were lysed with RIPA extraction reagent (Beyotime, China) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, USA). Total protein was separated using 10-12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 μm PVDF membrane (Millipore, USA). Primary antibodies were E-cadherin, N-cadherin, Vimentin, cyclin D1, CDK4, CDK6, GAPDH (Proteintech, Chicago), p-IRE1, XBP1s (Abcam, UK), AKT, or p-AKT (Cell Signaling Technologies, USA) antibodies. Bands were visualized using an enhanced chemiluminescence (ECL) kit (Boster, China) and detected by ChemiDoc XRS+ Imaging System (Bio-Rad).
10
Hippocampal Protein Analysis Protocol
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The ipsilateral hippocampus was isolated and kept at -80℃ immediately until use. The RIPA lysis buffer was employed to homogenize the and further span at 14,000 rpm at 4℃ for 30 minutes. We collected the supernatant and then employed the BCA Protein Assay Kit to quantify the proteins. The proteins were then fractionated on a 10% SDS-PAGE gel. After that, the fractionated proteins were blotted onto PVDF membranes. Then, 5% BSA was employed to block the membranes. After that, the membranes were inoculated with the primary antibodies consisting of; anti-GRP78 (1:500, Abcam, UK), anti-LC3B (1:500, Cell Signaling Technology, USA), p62 (1:500, Cell Signaling Technology, USA), IRE-1 (1:500, Abcam, UK), p-IRE-1 (1:500, Abcam, UK), JNK (1:500, Abcam, UK), p-JNK (1:500, Cell Signaling Technology, USA), beclin1 (1:500, Cell Signaling Technology, USA) overnight at 4°C. Thereafter, the membranes were inoculated with speci ed secondary anti-bodies for 2 hours at room temperature. Immunolabeling was done with the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scienti c, USA). The GE Amersham Imager 600 was employed to image the immunoblots and the Image J software utilized to analyze the protein bands. β-actin served as the normalization standard.
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