Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1S, 3R-RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700). PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti, 850375), PE (1-stearoyl-2-eicasotetraenoyl-sn-glycero-3-phosphatidylethanolamine, Avanti, 850804), PE-OOH (1-stearoyl-2-15-hydroperoxy-eicasotetraenoyl-sn-glycero-3-phosphatidylethanolamine, Cayman, Cay25856-100).
Anti calnexin
Manufactured by Proteintech
Sourced in United States
Anti-Calnexin is a primary antibody that recognizes the Calnexin protein. Calnexin is an endoplasmic reticulum resident chaperone protein that is involved in the quality control of newly synthesized glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti tsg101, by Proteintech (3 mentions)
Anti cd63, by Proteintech (3 mentions)
Anti flag, by Merck Group (2 mentions)
Anti tom20, by Proteintech (2 mentions)
Anti alix, by Proteintech (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti cd9, by Proteintech (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Lipofectamine ltx reagent with plus reagent, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose immunoprecipitation reagent, by Santa Cruz Biotechnology (1 mentions)
Liperfluo, by Dojindo Laboratories (1 mentions)
Anti glutathione peroxidase 4, by Abcam (1 mentions)
Anti gabarap, by Abcepta (1 mentions)
Fer 1, by Targetmol (1 mentions)
Pd146176, by Merck Group (1 mentions)
Zileuton, by Selleck Chemicals (1 mentions)
15 protocols using anti calnexin
1
Ferroptosis and Autophagy Assessment
2
Immunoblotting Protocol for Protein Detection
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For immunoblotting, samples resolved by SDS-PAGE were transferred onto 0.2 μm nitrocellulose membranes (Bio-Rad, cat# 1620112). Membranes were blocked with 5% non-fat milk, and then incubated with the appropriate primary antibodies: anti-HA (Sigma-Aldrich, cat# H3663), 1:5000; anti-Flag (Sigma-Aldrich, cat# F1804), 1:5000; anti-ICDH (Xu et al., 2010 (link)), 1:10,000; anti-tubulin (DSHB, E7) 1:10,000; anti-PDHA1 (ProteinTech, cat# 18068-1-AP), 1:5000; anti-ATPB (ProteinTech, cat# 17247-1-AP), 1:2000; anti-TOM20 (ProteinTech, cat# 11802-1-AP), 1:5000; anti-VDAC1 (ProteinTech, cat# 55259-1-AP), 1:2000; anti-Cyto c (Santa Cruz Biotechnology, cat# sc-13560), 1:1000; anti-Calnexin (ProteinTech, cat# 10427-2-AP), 1:2000; anti-ANT1/2 (ProteinTech, cat# 17796-1-AP), 1:1000; anti-GM130 (BD Biosciences, cat# 610822), 1:2500; and anti-ADPR (Sigma-Aldrich, cat# MABE1016), 1:1000. Membranes were then incubated with an appropriate IRDye infrared secondary antibody and scanned by using an Odyssey infrared imaging system (Li-Cor’s Biosciences).
3
Comprehensive Cell Line Characterization and Reagent Validation
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HeLa cells, B16‐F10, MDA‐MB‐231, Raw246.7, and HEK 293T cells were obtained from ATCC. 4T1 cells were kindly provided by Dr. Minh LE (National University of Singapore). All the cells were maintained in DMEM (Invitrogen, 12800‐017) containing 10% fetal bovine serum (Invitrogen, 10270‐106) and 100 units mL−1 of penicillin/streptomycin (Invitrogen, 15140‐122) at 5% CO2 and 37 °C. The following antibodies were purchased from Cell Signaling Technology (catalog numbers provided after each name): Anti‐Rab5 (3547), anti‐EEA1 (3288), anti‐LAMP1 (9091), anti‐Rab27a (69 295), anti‐Rab11 (5589), anti‐PD‐L1 (13684), anti‐granzymeB (17215), anti‐ATG5 (2630), and anti‐ATG4B (5299). Anti‐TSG101 (14497‐1‐AP), anti‐CD63 (25682‐1‐AP), anti‐Alix (12422‐1‐AP), anti‐Calnexin (10427‐2‐AP), anti‐GAPDH (60004‐1‐Ig), and anti‐Beta Actin (66009‐1‐Ig) antibodies were purchased from Proteintech. Anti‐PD‐L1 (MIH1, 16‐5983‐38), anti‐PD‐L1 (MIH5, 16‐5982‐38), anti‐CD3 (145‐2C11), and anti‐CD8 (56.6.7) antibodies, DQ‐green BSA (D12050), and Transferrin‐488 (T13342) were purchased from Thermo Fish Scientific. InVivoPlus Anti‐mouse PD1 (29F.1A12) antibody and InVivoPlus rat IgG2a isotype control (2A3) were purchased from Bio X Cell. GW4869 and Bafilomycin A1 were purchased from Selleck. Pipstop2 and Fillipin III were purchased from Cayman.
4
Characterizing Mitochondrial Dynamics Factors
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Anti-GFP (1:1000, 50430-2-AP, ProteinTech), anti-Mff (17090-1-AP; ProteinTech), anti-Drp1 (#8570; CST), anti-Mfn2 (12186-1-AP; ProteinTech), anti-PTPIP51 (20641-1-AP; ProteinTech), anti-calnexin (10427-1-AP; ProteinTech), anti-COXIV (11242-1-AP; ProteinTech), anti-flag (F1804; Sigma), and anti-p97/VCP (10736-1-AP; ProteinTech) were used at 1:1000 dilutions for Western blots. Anti-VAPB (14477-1-AP; ProteinTech) was used at 1:1000 for Western blots and 1:200 for immunofluorescence. Anti-tom20 (sc-17764; Santa Cruz Biotechnology), anti-tubulin (11224-1-AP; ProteinTech), and anti-actin (60008-1-Ig, ProteinTech) were used at 1:5000 dilutions for Western blots. Anti-VPS13D (A304-691A; BETHY Laboratories) was used at 1:50 dilutions for coimmunoprecipitation and 1:100 for Western blots. The reagents NMS873 (TargetMol, T1853) and BODIPY 493/503 (Thermo Fisher, D3922) were used. Antibiotics such as G418 (10131027), puromycin (A1113803), hygromycin B (10687010), and blasticidin (A1113903) were obtained from Thermo Fisher (U.S.).
5
Immunofluorescence Staining of Cellular Organelles
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After the indicated treatments as described in the figure legends, frozen slices or cells were fixed with 4% paraformaldehyde for 15 min at 37 °C and incubated with 0.2% Triton X100 for 15 min at room temperature. After blocking with 3% bovine serum albumin, the slices were incubated with primary antibodies. The following primary antibodies were used: anti-CD36 (#NB600-1423, Novus Biologicals), anti-SELK (#ab121276, Abcam), anti-SEC24 (#15958-1-AP, Proteintech), anti-His-Tag (#12698S, Cell Signaling Technology), anti-Calnexin (#66903-1-Ig, Proteintech), anti-GM130 (#11308-1-AP, Proteintech), anti-Golgin97 (#12640-1-AP, Proteintech), anti-TGN46 (#MA3-063, Invitrogen). After overnight incubation at 4 °C, the slices or cells were then incubated with fluorescence-conjugated secondary antibodies for 1 h. Finally, the slices or cells were incubated with DAPI for 3 min, and then, fluorescence images were captured using a Leica TCS SP8 confocal laser scanning microscope (Leica, Germany) and analyzed using Fiji ImageJ software.
6
Interrogating Regulated Cell Death Pathways
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Antibodies against MLKL (D6W1K) (#37705), MLKL (D2I6N) (#14993), Phospho-MLKL(Ser345) (D6E3G) (#37333), Phospho-MLKL(Ser358) (D6H3V) (#91689), RIP3 (E1Z1D) (#13526), XBP1 (E9V3E) (#40435), BIP (C50B12) (#3177), eIF2α (D7D3) (#5324), Phospho-eIF2α(Ser51) (D9G8) (#3398), Poly/Mono-ADP Ribose (E6F6A) (#83732), VDAC (D73D12) (#4866), GAPDH (14C10) (#2118) and β-Actin (13E5) (#4970) were purchased from Cell Signaling Technology. Anti-DFNA5/GSDME (#ab215191) and anti-AIF (#ab32516) were purchased from Abcam. Anti-Calnexin (#10427–1-AP) was purchased from Proteintech. Antibody against Histone H2A Rabbit mAb (#A3692) was purchased from ABClonal. Anti-mouse PD-1 (#BE0146) was purchased from BioXcell.
Sodium palmitate (#S161420) was purchased from Aladin. BSA (Fatty Acid & IgG Free) (#ST025) was purchased from Beyotime. Necrostatin-1 (#S8037), ferrostatin-1 (#S7243), Z-VAD-FMK (#S7023), Birinapant (#S7015), Olaparib (AZD2281) (#S1060), Ceapin-A7 (#E1099), GSK2606414 (#S7307), 4μ8C (HY-19707) (#S7272), and MNNG (#E0157) were obtained from Selleck Chemicals. SYTOX Green and Magnesium Green were purchased from Thermo Fisher Scientific. Recombinant murine and human TNF-α were purchased from Peprotech.
Sodium palmitate (#S161420) was purchased from Aladin. BSA (Fatty Acid & IgG Free) (#ST025) was purchased from Beyotime. Necrostatin-1 (#S8037), ferrostatin-1 (#S7243), Z-VAD-FMK (#S7023), Birinapant (#S7015), Olaparib (AZD2281) (#S1060), Ceapin-A7 (#E1099), GSK2606414 (#S7307), 4μ8C (HY-19707) (#S7272), and MNNG (#E0157) were obtained from Selleck Chemicals. SYTOX Green and Magnesium Green were purchased from Thermo Fisher Scientific. Recombinant murine and human TNF-α were purchased from Peprotech.
7
Exosome Protein Expression Analysis
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In brief, the exosomes and collected cells were lysed with a RIPA lysis buffer (Beyotime, Shanghai, China) to extract the proteins. The total protein concentration was detected using the BCA Protein Assay kit (Beyotime, Shanghai, China). Then, the protein was separated using SDS-PAGE and was transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 h. Next, the membrane was incubated with primary antibodies: anti-CD63 (Santa Cruz, Dallas, TX, USA), anti-TSG101 (Santa Cruz, Dallas, TX, USA), anti-HSP70 (Santa Cruz, Dallas, TX, USA), anti-Calnexin (Proteintech, Chicago, IL, USA), anti-JNK (Abcam, Cambridge, UK), anti-P-JNK (Abcam, Cambridge, UK), anti-CD36 (Proteintech, Chicago, IL, USA), anti-SIRP-α (Proteintech, Chicago, IL, USA), and anti-IL-4R (Proteintech, Chicago, IL, USA) at 4 °C overnight. After washing with a TBST buffer three times, the membrane was incubated with the secondary antibody at room temperature for 90 min. Finally, the protein bands were visualized with enhanced chemiluminescence (Thermo Fisher, Waltham, MA, USA). The immunoreactive bands were quantified with Image J software (Version 1.8.0, National Institutes of Health, Bethesda, MD, USA).
8
Comprehensive Characterization of hiPSC-Exosomes
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The protein concentration of hiPSC-exosomes was determined using a BCA protein quantitation kit (Zoman Biotechnology, ZD301). An HT7700 transmission electron microscope (Hitachi, Japan) was used to determine the morphology and size of hiPSC-exosomes. The particle size distribution of hiPSC-exosomes was measured by nanoparticle tracking analysis (Particle Metrix zataview, Germany). Marker proteins of hiPSC-exosomes were detected by western blotting using anti-CD9 (Yang et al., 2022 (link)) (Affinity, DF6565), anti-TSG101 (Qi et al., 2022 (link)) (Proteintech, 67381-1-Ig), and negative protein marker anti-Calnexin (Sun et al., 2021a (link)) (Proteintech, 66903-1-Ig) antibodies. hiPSC-exosome-specific protein markers CD63 (BD, 556019) and CD81 (BD, 551108) were detected by a Flow NanoAnalyzer (Nanofcm, China).
9
Western Blot and ELISA Analysis of Exosomal Proteins
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For western blot, proteins from cells or exosomes were loaded and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies at 4°C overnight, including anti-collagen I, anti-AT1R, anti-Alix, anti-CD9, anti-CD63, anti-calnexin (1:1000, Proteintech, Wuhan, China), anti-TGF-β, anti-p-Smad2/3, anti-Smad2/3 (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:7500, Ray Antibody Biotech, Beijing, China), and then incubated with anti-rabbit or anti-mouse near-infrared secondary antibodies (1:15,000, LI-COR, Inc., Lincoln, UK) for 1 h. The membrane was exposed to Odyssey® CLx Imager (LI-COR), and Odyssey Software (LI-COR) was used for capturing images and the data analysis.
Cell culture supernatants, BALF, and serum were analyzed for Ang II using Ang II ELISA kits (Suzhou Calvin Biotechnology Co., Ltd, Suzhou, China), according to the manufacturer's instructions.
Cell culture supernatants, BALF, and serum were analyzed for Ang II using Ang II ELISA kits (Suzhou Calvin Biotechnology Co., Ltd, Suzhou, China), according to the manufacturer's instructions.
10
Quantitative Western Blot Protocol
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Western blot was performed as previously described (Wang et al., 2019 (link)). Briefly, whole cell extracts were prepared using lysis buffer (50 mM Tris–HCl, 0.5% (vol/vol) NP-40, 1% Triton-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% protease inhibitor cocktails, 1 mM Na3VO4, and 1 mM NaF, pH 7.4). Proteins were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes or PVDF membranes. The membranes were incubated in 0.1% PBST with 5% BSA, followed by PBS washing and primary antibody incubation at 4°C overnight. The primary antibodies included: anti-ZIKV envelope (E) (GeneTex, GTX 133314), anti-p62 (Santa Cruz, sc-28359), anti-GRP78 (Proteintech, 11587-1-AP), anti-Calnexin (Proteintech, 66903-1-lg), anti-FLAG (MBL, PM020), and anti-anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 10494-1-AP). Detection was performed with IRDye 800 CW-conjugated anti-rabbit IgG and IRDye 680 CW-conjugated anti-mouse IgG secondary antibody (LI-COR) according to the manufacturer’s protocols. Immunoreactive bands were visualized using an Odyssey infrared imaging system (LI-COR) as described previously (Doms et al., 2018 (link)).The western blot bands were quantified by Quantity One (Bio-Rad).
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