The 7S glycinin and 11S glycinin were provided by Professor Shuntang Guo of China Agricultural University (patent number: 200 410 029 589.4, Beijing, China). Primary antibodies against phosphorylated IRE1α (p-IRE1α; #ab48187), glucose-regulated protein 78 (GRP78; #ab108615), recombinant apoptosis signal-regulating kinase 1 (ASK1, #ab278547), recombinant activating transcription factor 6 (ATF6; #ab37149), and glyceraldehyde phosphate dehydrogenase (GAPDH; #ab9485) were obtained from Abcam (Cambridge, UK). Primary antibodies against C/EBP-homologous protein (CHOP; #mAb2895), LC3 (#mAb12741), sequestosome-1 (p62; #mAb39749), and XBP-1s (#mAb40435) were purchased from Absin Bioscience Inc. (Shanghai, China). Primary antibodies against phospho-protein kinase R-like ER kinase T982 (p-PERK; #AP0886), enzyme-linked immunosorbent assay (ELISA) kits for histamine (HIS), immunoglobulin E (IgE), 5-hydroxytryptamine (5-HT), IL-18, IL-1β, D-lactic acid (D-LA), diamine oxidase (DAO), and secreted mucin-2 (MUC-2) were purchased from Shanghai Youxuan Biotechnology Co., Ltd. (Shanghai, China). The PrimeScript™ RT Reagent (TRIzol) kit was obtained from Takara (Beijing, China).
Ab37149
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab37149 is a laboratory equipment product. It is a core-shell silica-based stationary phase designed for high-performance liquid chromatography (HPLC) applications. The product specifications and details are not available for an unbiased and factual description without interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab48187, by Abcam (6 mentions)
Ab37073, by Abcam (4 mentions)
Ab21685, by Abcam (4 mentions)
Ab37152, by Abcam (3 mentions)
Ab203119, by Abcam (2 mentions)
Ab229912, by Abcam (2 mentions)
Ab124956, by Abcam (2 mentions)
Ab11419, by Abcam (2 mentions)
Sc 376768, by Santa Cruz Biotechnology (2 mentions)
Ab108615, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Gtx109639, by GeneTex (1 mentions)
Df7576, by Affinity Biosciences (1 mentions)
Gsk2656157, by MedChemExpress (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Tunicamycin, by MedChemExpress (1 mentions)
Pepsin, by Merck Group (1 mentions)
Ab34712, by Abcam (1 mentions)
Anti phospho stat1 antibody, by Cell Signaling Technology (1 mentions)
16 protocols using ab37149
1
Glycinin Extraction and Characterization
2
ER Stress Pathway Molecular Markers
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The primary antibodies used are listed as follows: anti-cytochrome c (1:1,000, 10993-1-AP, Proteintech, rosemont, IL, USA), anti-caspase-9 (1:1,000, #9508, Cell Signaling, Danvers, MA, USA), anti-PERK (1:1,000, #3192, Cell Signaling, Danvers, MA, USA), anti-phosphorylated-PERK (anti-p-PERK; Thr982;1:800, DF7576, Affinity Biosciences, Cincinnati, OH, USA), anti-IRE1 (1:500, ab37073, Abcam, Cambridge, MA, USA), anti-p-IRE1 (phosphor S724; 1:1,000, ab48187, Abcam, Cambridge, MA, USA), anti-activating transcription factor 6 (anti-ATF6; 1:1,000, ab203119, Abcam, Cambridge, MA, USA), anti-N’ATF6 (1:800, ab37149, Abcam, Cambridge, MA, USA) and anti-β-actin (1:3,000, GTX109639, GeneTex, Irvine, CA, USA). The ER stress activator Tunicamycin (TM) and the p-PERK inhibitor GSK2656157 were obtained from MedChem Express (Monmouth Junction, NJ, USA). The p-IRE1 inhibitor 3,5-dibromosalicylaldehyde (DBSA) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan).
3
Immunofluorescence Characterization of Cartilage Collagens
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Immunofluorescence on tibial cryosections was essentially performed as previously described [30 ]. The primary antibodies used were anti-COL2A1 (Abcam, ab34712, 1:100), anti-COL10A1 (from Kathryn Song Eng Cheah, University of Hong Kong, 1:500), anti-COL9A1 (Bioworld Technology, BS6945, 1:50), anti-COL11A1 antibody (Abcam, ab64883, 1:100), anti-ATF6 antibody (Abcam, ab37149, 1:100), anti-ATF4 antibody (Abcam, ab31390, 1:100), anti-GRP78 BiP antibody (Abcam, ab21685, 1:100), anti-XBP1 antibody (Abcam, ab37152, 1:100), anti-phospho- Erk1/2 antibody (Cell Signaling Technology, 4370, 1:200), anti-phospho-Stat1 antibody (Cell Signaling Technology, 9167, 1:200). Alexa Fluor 594 conjugate of Concanavalin A (Invitrogen) was used to colocalized with collagens. For ColII/IX/XI antigen retrieval, sections were treated with 2 mg/ml pepsin (Sigma Aldrich) for 10 min at 37 °C. For ColX antigen retrieval, sections were digested with 2 mg/ml hyaluronidase (Sigma Aldrich) for 20 min at 37 °C. The primary antibodies were detected with appropriate Alexa Fluor-conjugated secondary antibodies (Abcam). All sections were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen) and visualized using a fluorescent microscope (Zeiss).
4
Comprehensive Immunoblotting Antibody Panel
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The following primary antibodies were used: mouse monoclonal antibodies against MHV N and S proteins (kind gifts of Dr Helmut Wege, University of Würzburg), rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (ab272504, Abcam) mouse anti-GAPDH (IgM specific, G8795, Sigma-Aldrich), mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-HA (3724, Cell Signaling Technology), rabbit anti-PERK (ab229912, Abcam), rabbit anti-HERPUD1 (ab150424, Abcam), rabbit anti-GRP78 (BIP, ab108613, Abcam), rabbit anti-eIF2α (9722, Cell Signaling Technology), rabbit anti-phospho-eIF2α (Ser51, 9721, Cell Signaling Technology), rabbit anti-ATF4 (10835-1-AP, Proteintech), rabbit anti-ATF6 (ab203119 and ab37149, Abcam), mouse anti-S6 (2317, Cell Signaling Technology) and rabbit RPL10a (ab174318, Abcam). Secondary antibodies used for western blotting were purchased from Licor: IRDye 800CW Donkey Anti-Mouse IgG (H+L), IRDye 800CW Donkey Anti-Rabbit IgG (H+L), IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 680RD Goat Anti-Mouse IgM (μ chain specific).
5
Quantifying Cellular ER Stress Markers
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Human osteosarcoma U2OS cells were treated for 6 h to detect eIF2α phosphorylation (PeIF2α) and TFE3, 16 h to assess ATF6 and spliced XBP1 (XBP1s) levels, or 24 h to measure CHOP expression. Then cells were fixed by 3.7% PFA at 4 °C overnight. For staining, fixed cells were then permeabilized with 0.1% Triton X100 on ice, and blocked with 5% bovine serum albumin (BSA, w/v in PBS) for 1 h. Next, cells were incubated with antibodies specific to TFE3 (#ab93808, 1:400, Abcam), phospho-eIF2 alpha (Ser51) (#ab32157, 1:1000, Abcam), ATF6 (#ab37149, 1:200, Abcam), XBP1 (#ab37152, 1:250, Abcam) or CHOP (#2895, 1:500, Cell Signaling Technology) at 4 °C overnight. After washed by PBS twice, AlexaFluor conjugates (Thermo Fisher Scientific) against the primary antibody were applied for 2 h at RT. Finally, cells were washed and imaged by automated fluorescence microscopy as described above. The nuclear intensity of TFE3, ATF6, XBP1s or CHOP and cytoplasmic intensity of phospho-eIF2α (Ser51) were measured and normalized on Ctrl.
6
Endoplasmic Reticulum Stress Pathway
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Antibodies against phosphorylated (p)-protein kinase R-like ER kinase (PERK; ab192591), PERK (ab79483), inositol-requiring kinase 1α (IRE1α; ab37073), p-IRE1α (ab48187), activating transcription factor 6α (ATF6α), p-50ATF6α (ab37149), glucose-regulated protein 78 (GRP78; ab21685), CCAAT/enhancer binding protein homologous protein (CHOP; ab11419), p-c-Jun N-terminal kinase (JNK; ab124956), JNK (ab179461) and caspase-3 (ab13847) were purchased from Abcam (Cambridge, UK). N-acetyl L-cysteine (NAC), 4-phenylbutyric acid (4-PBA) and thap-sigargin (Thap) were also purchased from Abcam. The Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from Roche Diagnostics (Indianapolis, IN, USA). Cell Counting kit-8 (CCK-8) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). SW (osmolality, 1,300 mmol/l; pH 8.2; relative density 1.05; salt content, 34.421%; NaCl, 26.518 g/l; MgSO4, 3.305 g/l; MgCl2, 2.447 g/l; CaCl2, 1.141 g/l; KCl, 0.725 g/l; NaHCO3, 0.202 g/l; and NaBr, 0.083 g/l) was prepared based on the overall composition of the East China Sea, which was provided by the Chinese Ocean Bureau (Beijing, China).
7
Western Blot Analysis of Retinal Proteins
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Total cellular and nuclear protein was extracted from retinas and quantified using bicinchoninic acid assay kit. Homogenate in 2 × sodium dodecyl sulfate (SDS) sample buffer was boiled for 5 min, and then equal amounts of protein (40 μg) from each sample were subjected to electrophoresis on a 10% (v/v) SDS-polyacrylamide gel. After proteins were electroblotted to a polyvinylidene difluoride membrane, the membrane was blocked with Phosphate-buffered saline containing 5% dried non-fat milk or 3% BSA at room temperature for 1 h, and incubated with indicated primary antibodies (anti-ATF4, 1:1000, SC-200, Santa Cruz Biotechnology, Dallas, TX; anti-ATF6, 1:1500, ab37149; anti-GRP78/BiP, 1:2000, ab21685; anti-pIRE1α, 1:1000, ab48187, Abcam, Cambrigde, MA; anti-Nrf2, 1:1000, SC-722; Santa Cruz Biotechnology, Dallas, TX; anti-p38, 1:500, ab27986; anti-p-p38, 1:1000, ab4822; anti-JNK, 1:1000, ab59227; anti-p-JNK, 1:2000, ab124956; anti-p65, 1:600, ab7970; anti-p-p65, 1:2000, ab86299; anti-SIRT1, 1:2000, ab12193, Abcam, Cambrigde, MA) at 4 °C overnight, followed by incubating with the goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody for 2 h. After incubation, membrane was washed three times, and the antigen-antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer, Akron, OH).
8
Validating Anti-ATF6 Antibody Using Colocalization Assay
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To validate anti-ATF6 antibody (ab37149; abcam, Cambridge, MA, USA), we performed a colocalization assay using flag-tagged ATF6-transfected COS-7 cells. The cells were grown on coverslips in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10 % fetal bovine serum and then transfected with Lipofectamine reagent (Invitrogen). After incubation at 37 °C with 5 % CO2 for 24 h, transfected cells were fixed with 4 % paraformaldehyde (EMS) in PBS for 30 min, permeabilized for 15 min in 0.2 % Triton X-100 and nonspecific binding sites were blocked by incubation in 2 % BSA and 5 % normal goat serum in PBS for 30 min. Primary ATF6 and anti-flag antibodies were diluted in blocking solution to a final concentration of ~0.5 μg per ml. COS-7 cells were incubated for 2 h at room temperature with ATF6 and anti-flag antibodies, followed by incubation with Alexa Fluor 546 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes) for 45 min at room temperature. Rhodamine phalloidin labeled the actin cytoskeleton and DAPI was used to stain nuclei. Samples were washed in PBS, mounted using ProLong Gold Antifade reagent (Molecular Probes), and imaged with the use of a LSM510 coreduo confocal microscope.
9
Liver Tissue Protein Analysis Protocol
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Qiagen Tissue Homogenizer was used to homogenize the liver tissues (1 min, on ice). Cultured hepatocytes were harvested in Cell Lysis Buffer (Cell Signaling Technology). Cellular lysates were sonicated for 2 min and immunoblotted to examine the target proteins with antibodies against XBP1 (SC-7160, Santa Cruz, or #12782, Cell Signaling), phospho-AKTS473 (#9271), phospho-GSK3βS9 (#9336), phospho-MEK1/2S217/221 (#9154), phospho-ERKT202/204 (#4370), phospho-PERKT980 (#3179), phospho-eIF2αS51(#9721), phospho-p38T108/182 (#4631), eIF2α (#9722), IRΕ1 (#3294) (Cell signaling), and phospho-IRE1S724 (ab48187), ATF6 (ab37149), G6pc (ab83690), and PEPCK (ab70358) (abcam) at concentrations recommended by the manufacturers. Secondary antibodies were used at the concentrations around 1:5000 ∼ 10,000.
10
Western Blot Analysis of ER Stress Markers
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Total proteins were extracted from 3T3-L1 cells using RIPA buffer (Biomax, Seoul, Korea) and a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The cell extract proteins were then separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with one of the following primary antibodies: anti-ATF6 (1:1,000, AB37149, Abcam, Cambridge, UK), anti-phospho-PERK (1:1,000, SAB5700521, Sigma), anti-PERK (1:1,000, C33E10, Cell Signaling, Danvers, MA, USA), anti-phospho-eIF2α (1:1,000, 9721, Cell Signaling), anti-eIF2α (1:1,000, 9722, Cell Signaling), or anti-β-actin (1:2,000, D6A8, Cell Signaling). Subsequently, each membrane was washed with TBS with 0.05% Tween-20 and incubated at room temperature with a horseradish peroxidase-linked rabbit secondary antibody (Cell Signaling, 7074) for 1 h. After incubation, membranes were stripped using a stripping buffer (Biomax). Fluorescent signals were visualized using West Glow FEMTO chemiluminescent substrate (Biomax) and were quantified using ImageJ software.
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