Tissue sections were blocked with 3% BSA for 30 min at 25 °C. Sections were incubated overnight at 4 °C with antibodies against integrin αVβ5 (1:100, Santa Cruz Biotechnology, Japan). For GC and TC staining, sections were fixed in 4% paraformaldehyde (Servicebio, China) for 30 min at 25 °C and then permeabilized with 0.3% Triton X-100 (Beyotime, China). After washing with PBS three times, the cells were blocked with 3% BSA for 30 min at 25 °C. Cells were incubated with antibodies against IRE1α (1:100, Proteintech), apoptosis-associated speck-like protein containing a CARD (ASC, 1:100, AdipoGen Life Science, USA), NLRP3 (1:100, CST, USA), a-smooth muscle actin (α-SMA, 1:100, Abcam, UK) and collagen I (1:100, Bioworld Technology, China) overnight at 4 °C. After washing with PBS three times, tissue sections and cells were incubated at 25 °C for 2 h with fluorescent secondary antibodies (Beyotime). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime) at a dilution of 1:2000 for 30 min and photographed using an Olympus laser scanning confocal microscope (FV3000, Japan).
Ire1α
Manufactured by Proteintech
Sourced in United States
IRE1α is a protein that functions as a sensor of endoplasmic reticulum (ER) stress. It is a key component of the unfolded protein response (UPR) pathway, which is a cellular mechanism that helps maintain ER homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Ire1α, by Cell Signaling Technology (2 mentions)
Triton x 100, by Beyotime (1 mentions)
Fluorescent secondary antibody, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Collagen 1, by Bioworld Technology (1 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Annexin 5 pi staining kit, by Thermo Fisher Scientific (1 mentions)
Tunicamycin, by MedChemExpress (1 mentions)
Imatinib, by MedChemExpress (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mkc8866, by MedChemExpress (1 mentions)
Metformin, by MedChemExpress (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ppp1cc, by Proteintech (1 mentions)
Mef2c, by Proteintech (1 mentions)
Eif2α, by Thermo Fisher Scientific (1 mentions)
Nanog, by Proteintech (1 mentions)
C myc, by Proteintech (1 mentions)
8 protocols using ire1α
1
Integrin and Inflammasome Immunostaining
2
Imatinib, Metformin, Tunicamycin, and MKC8866 Cytotoxicity
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Imatinib (HY-15463) and metformin (HY-B0627) and tunicamycin (HY-A0098), MKC8866 (HY-104040) were obtained from MedChemExpress (MCE). Annexin V/PI staining kit was purchased from Thermo Fisher Scientific (BMS500FI-100). Antibodies against the following proteins were used: CD34 (Abcam, ab81289), IRE1α (endoribonuclease α, Proteintech, 27528-1-AP), PERK (Protein kinase R-like endoplasmic reticulum kinase, Proteintech, 24390-1-AP), Phospho-PERK (p-PERK, Thr982, Beyotime Biotechnology, AF5902), and β-actin (Cell Signaling Technology, #4970).
3
Immunofluorescent Analysis of ER Stress
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Capan-2 and SW1990 cell lines were implanted into 24-well culture plates covered with slices, fixed in 4% paraformal dehyde, permeabilized with Triton X-100 (0.5%) and incubated with 5% BSA. Then plates were incubated with the primary antibodies overnight: CRT (Abcam) and IRE1α (Cell signaling technology). The secondary antibodies (Proteintech, Chicago, IL, USA) were conjugated with FITC for CRT and TRITC for IRE1α. Hoechest33258 were used for nuclear visualizing.
4
Protein Expression Analysis in Muscle Tissue
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Tissue was homogenized and then lysed in ice-cold RIPA buffer (50mM of Tris–HCl (pH 7.4), 150mM of NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 2 mM of EDTA, and 1× protease inhibitor cocktail). Samples were then centrifuged for 20 min at 4°C. Total protein extracts were loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to nitrocellulose membranes (Whatman). Membranes were then blocked with 5% milk for 1 h. Membranes were incubated with primary antibodies against MYOD (Proteintech Cat# 18943-1-AP), MEF2C (Proteintech Cat# 10056-1-AP), MyHC (R&D Cat# MAB4470), PERK (CST Cat# 3192), p-PERK (CST Cat# 3179), phospho-eIF2α (1:1000, CST Cat# 3398), eIF2α (CST Cat# 9722), ATF4 (CST Cat# 11815), p-ATF4(Ser219) (Thermo Fisher Cat# PA5-105835), ATF6 (CST Cat# 65880), IRE1α(CST Cat# 3294), PPP1CC (Proteintech Cat# 55150-1-AP), ARPP21 (Proteintech Cat# 55150-1-AP), c-Myc (CST Cat# 5605), KLF4 (CST Cat# 4038), Nanog (CST Cat# 8822), SOX2 (CST Cat# 4962), OCT4 (CST Cat# 2840), PAX7 (Proteintech Cat# 20570-1-AP), and GAPDH (Proteintech Cat# 55150-1-AP) overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies were used to detect the primary antibodies and protein signals were then visualized using Chemiluminescent HRP Substrate (Millipore, WBKLS0500).
5
Hepatic Fibrosis Pathway Regulation
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CPT (purity ≥ 98% purity by HPLC, Cat No. HY-N0174), were purchased from Med Chem Express LLC (Shanghai, China) (Figure 1A ). Primary antibodies for α-SMA, Collagen I, eIF2α, p-eIF2α, p-PERK, PERK, IRE1-α, BAX, BCL2, CHOP, and GRP78/BIP and all secondary antibodies were purchased from Proteintech (China). ATF4, and XBP1 were purchased from Sigma (United States). The primers used in quantitative reverse transcription polymerase chain reaction (qRT-PCR) were purchased from Qingke Co. Ltd. (China) (Table 1 ).
6
Immunofluorescence Analysis of IRE1α and TRAF2
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Paraffin-embedded liver sections were deparaffinized with xylene and rehydrated with gradient ethanol. Sections were processed with EDTA antigen retrieval buffer (pH 8.0), 3% hydrogen peroxide (H2O2), and goat serum. Then, sections were incubated with antibodies IRE1α (Proteintech, 27528-1-AP, Wuhan, China), HRP goat anti-rabbit secondary antibodies (Servicebio, GB23303, Wuhan, China), and CY3-TSA (Servicebio, G1223, Wuhan, China). Next, sections were immersed in sodium citrate antigen retrieval buffer (pH 6.0). Additionally, sections were incubated with antibodies TRAF2 (Abcam, ab126758, Cambridge, UK) and secondary antibodies. Then, the nucleus was redyed with DAPI (Servicebio, G1012, Wuhan, China). Finally, sections were processed with spontaneous fluorescence quenching reagent and anti-fade mounting medium. The IF staining results were analyzed by Image-Pro Plus 6.0 software.
7
Immunostaining of ER Stress Markers
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Capan-2 and SW1990 cell lines were implanted into 24-well culture plates covered with slices, xed in 4% paraformal dehyde, permeabilized with Triton X-100 (0.5%) and incubated with 5% BSA. Then plates were incubated with the primary antibodies overnight: CRT (Abcam) and IRE1α (Cell signaling technology). The secondary antibodies (Proteintech, Chicago, IL, USA) were conjugated with FITC for CRT and TRITC for IRE1α. Hoechest33258 were used for nuclear visualizing.
8
Hippocampal Protein Profiling in IVH
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Mice were euthanized after IVH (n = 6) at experimentally appropriate time, and total proteins were harvested from the hippocampus in radioimmunoprecipitation assay (RIPA) lysis buffer. We transferred the proteins to polyvinylidene uoride membranes (Millipore), which were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with following primary antibodies at 4℃:anti-bodies against GSDMDC1(1:100; Santa Cruz, sc-395381), NLRP3(1:200; Santa Cruz, sc-134306), IL-1β(1:1000; Abcam, ab254306), Cleaved Caspase-1(1:1000; Cell Sign Technology, 4199), ASC(1:1000; Cell Sign Technology, 67824), CHOP(1:1000; Cell Sign Technology, 2895), BIP(1:1000; Cell Sign Technology, 3177), ATF-4(1:1000; Cell Sign Technology, 11815) and IRE1α(1:1000; proteintech, 27528). After that, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1h at room temperature. Finally, the results were quanti ed by Image J (NIH, USA).
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