Cells were solubilized with radioimmunoprecipitation assay (RIPA) buffer (Tris-HCl (50 mM), pH 8.0; NaCl (150 mM); NP-40 (1%); sodium deoxycholate (0.5%); and SDS (0.1%)) and protease inhibitor cocktail (Promega, Madison, WI, USA). The primary antibodies were as follows: rabbit anti-CYLD (1:250; Sigma-Aldrich, Cat. No. SAB4200060), mouse anti-GADD153 (B-3) (1:200; Santa Cruz Biotechnology, Cat. No. sc-7351), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; Medical & Biological Laboratories), mouse anti-BiP/GRP78 (1:250; BD Biosciences, San Jose, USA), and mouse anti-β-actin antibody (1: 10,000; Sigma-Aldrich). After incubation with the primary antibodies for 16 h at 4 °C, the membranes were reacted with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) for 1 h. Bands were detected by ECL prime western blotting detection system (GE Healthcare) with ImageQuant LAS-4000 mini EPUV (Fuji film, Tokyo, Japan). Grouping of blots were cropped from different parts of the same gel, and intensity of bands was quantified by ImageJ 1.50i software (National Institutes of Health, NIH).
Mouse anti bip grp78
Manufactured by BD
Sourced in United States
Mouse anti-BiP/GRP78 is an antibody that specifically binds to the BiP/GRP78 protein, a member of the heat shock protein 70 (Hsp70) family. BiP/GRP78 is a key regulator of the unfolded protein response and plays a critical role in protein folding and quality control within the endoplasmic reticulum.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Promega (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Ecl prime western blotting detection system, by GE Healthcare (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti phospho p38 mapk t180 y182, by Cell Signaling Technology (1 mentions)
Hypersensitive ecl chemiluminescence kit, by Beyotime (1 mentions)
Rabbit anti actin, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Rabbit anti slug, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Mouse anti zo 1, by Thermo Fisher Scientific (1 mentions)
Mouse anti n cadherin, by BD (1 mentions)
Rabbit anti cd9, by Abcam (1 mentions)
Rabbit anti vimentin, by GeneTex (1 mentions)
Mouse anti cd63, by Abcam (1 mentions)
Mouse anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
4 protocols using mouse anti bip grp78
1
Western Blot Analysis of CYLD, GADD153, and BiP
2
Western Blot Analysis of Cellular Proteins
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Total cellular proteins were extracted with the RIPA lysis buffer (Beyotime, Shanghai, China). Fifty micrograms (50 μg) of total protein lysate was separated using a 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), which were blocked with 5% skimmed milk for 1 h at room temperature. The membrane was incubated with primary antibodies overnight at 4°C and subsequently incubated with horseradish peroxidase-labeled secondary antibodies against rabbit/mouse IgG (Sigma-Aldrich, St. Louis, Missouri, USA) for 1 h at room temperature. We used the following primary antibodies: rabbit anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-Bip/GRP78 (BD Biosciences, Bedford, MA), mouse anti-myc (Santa Cruz, CA, USA), mouse anti-ATF6/cleaved ATF6, and rabbit anti-phospho-p38 MAPK (T180/Y182) (Cell Signaling, Beverly, MA). The target bands were visualized with a hypersensitive ECL chemiluminescence kit (Beyotime, Shanghai, China). ImageJ software was used for densitometric analysis.
3
Exosome Protein Profiling by Western Blot
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Proteins were extracted from exosome and cell lysates, using radio immunoprecipitation (RIPA) buffer containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Burlington, Massahusetts, USA). The membranes were blocked with 5% non-fat milk in Tris-buffer saline and 0.1% Tween-20 and probed with the following primary antibodies: mouse anti-CD63 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-CD9 (1:2000, Abcam), mouse anti-CD81 (1:250, Invitrogen), and mouse anti-Bip/Grp78 (1:1000, BD Biosciences, San Jose, CA, USA), rabbit anti-ENG (1:1000, Abcam), rabbit anti-SEC14L2 (1:1000, Abcam), mouse anti-ZO-1 (1:1000, Thermo Fisher Scientific), mouse anti-N-cadherin (1:2000, BD bioscience), rabbit anti-Vimentin (1:5000, GeneTex, Alton, CA, USA), rabbit anti-Slug (1:1000, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Chemiluminescence signals were detected using Clarity™ Western ECL Substrate and ChemiDoc (both from Bio-Rad Laboratories, Hercules, CA, USA).
4
Antibody Validation and siRNA Screening
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Antibodies to human Sec61β (1:10,000 dilution), TRAPα (ref. 49 (link); 1:5,000) and TMCO1 (ref. 9 (link); 1:5,000) were described previously. Other antibodies were obtained from the following commercial sources: rabbit anti-nicalin (A305-623A-M; 1:1,000) and rabbit anti-CCDC47 (A305-100A; 1:2,000) from Bethyl Laboratories; mouse anti-HA (326700; 1:1,000), goat anti-NOMO (PA5-47534; 1:1,000), rabbit anti-TMEM147 (PA5-95876; 1:1,000), rabbit anti-Asterix (PA5-66788; 1:5,000), rabbit anti-C20Orf24 (PA5-43332; 1:1,000) and rabbit anti-Sec61α (PA5-21773; 1:1,000) from Invitrogen; rabbit anti-uL22 from Abgent (AP9892b; 1:1,000); mouse anti-tubulin (ab11304; 1:1,000) and mouse anti-HRP (ab6728; 1:1,000) from Abcam; mouse anti-Flag (F1804; 1:1,000), rabbit anti-Flag (F7425; 1:1,000), rabbit anti-peroxidase (SAB3700863; 1:10,000) and goat anti-peroxidase (A5420; 1:20,000) from Sigma; mouse anti-STT3A (H00003703-M02; 1:1,000) from Novus Biologicals; mouse anti-BiP/GRP78 (610979; 1:1,000) from BD Biosciences. siRNAs were purchased from Thermo Fisher: negative control (4390843), TMCO1 (s29085), C20Orf24 (s31821), Asterix (s28089), CCDC47 (s32576), Nicalin (s32411) and TMEM147 (s20404).
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