Grp78

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom

GRP78 is a molecular chaperone protein that plays a crucial role in the endoplasmic reticulum (ER) of eukaryotic cells. It is involved in the folding and assembly of proteins within the ER, helping to maintain ER homeostasis and prevent the accumulation of misfolded proteins.

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148 protocols using grp78

Comprehensive Protein Detection Techniques

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For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, ♯sc-47778; 1:5000), ATF6 (Cell Signaling Technology, ♯65880; 1:1000), BCL-2 (AbCam, ♯ab182858; 1:500), BCL-XL (Cell Signaling Technology, ♯2764; 1:1000), Cleaved-Caspase-8 (Cell Signaling Technology, ♯8592; 1:1000), Caspase-3 (Cell Signaling Technology, ♯9665; 1:1000), Caspase-9 (Cell Signaling Technology, ♯9508; 1:1000), CHOP (Cell Signaling Technology, ♯2895; 1:250), GFP (AbCam, ♯ab13970; 1:2000), GRP78 (Cell Signaling Technology, ♯3177; 1:1000), FLAG-HRP (Sigma Aldrich, ♯A8592; 1:4000), HA-HRP (Roche, ♯11867423001; 1:2000), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:1000), IP₃R1 & IP₃R2 [previously described⁷⁷; 1:1000], IP₃R3 (BD Biosciences, ♯610312; 1:1000), KDEL (AbCam, ♯ab12223; 1:2000), MCL-1 (Cell Signaling Technology, ♯5453; 1:1000), Nicastrin (BD Biosciences, ♯612290; 1:1000), PARP (Cell Signaling Technology, ♯9542; 1:1000), SERCA2 (Cell Signaling Technology, ♯4388; 1:1000), STIM1 (Cell Signaling Technology, ♯5668; 1:1000).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:200), IP₃R3 (BD Biosciences, ♯610312; 1:200).

Dulloo I., Atakpa-Adaji P., Yeh Y.C., Levet C., Muliyil S., Lu F., Taylor C.W, & Freeman M. (2022). iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2. Nature Communications, 13, 1257.

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Antibody Procurement and Cell Assays

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Antibodies against ATF6, CIP2A, GRP78, GAPDH, and β‐Actin were purchased from Cell Signaling Technology (Boston, MA, USA). Antibody against CIP2A for immunohistochemistry (IHC) was purchased from Novus Biologicals (Littleton, CO, USA), and for western blot analysis was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody against ERK was purchased from Zymed (Grand Island, NY, USA). Tunicamycin, cycloheximide, 3‐[4, 5‐dimethylthiazol‐ 2‐y1]‐2, 5‐diphenyltetrazolium bromide) (MTT), and 4‐(2‐Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

Liu C., Hsu C., Huang T., Lee C., Chen J., Yang S., Jiang J., Chen W., Lee K, & Teng H. (2018). ER stress‐related ATF6 upregulates CIP2A and contributes to poor prognosis of colon cancer. Molecular Oncology, 12(10), 1706-1717.

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Western Blot Analysis of UPR Markers

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Caco-2 monolayers were lysed directly in Laemmli buffer supplemented with 5% (v/v) 2-mercaptoethanol and boiled for 15 minutes. Equal amounts of lysate were separated by electrophoresis on SDS-polyacrylamide gels (Biorad) under denaturing conditions. Proteins were transferred to PVDF membranes (Millipore) via wet electrotransfer. After protein blotting, membranes were blocked in PBS with 0.1% (v/v) Tween-20 with either 5% (w/v) non-fat dry milk or 5% (w/v) BSA. Primary antibodies against active Caspase-3 (BD Biosciences), CHOP (Cell Signalling), GRP78 (Cell Signalling), XBP1s (BioLegend), or βActin (Santa Cruz) were applied overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies raised against mouse, rabbit or goat immunoglobulins (Abcam). Protein bands were visualized with ECL (Pierce) and ChemiDoc MP System (Bio-Rad). Quantification of the bands were performed by imageJ software, with the intensity values normalized to the corresponding βActin band.

Akiyama T., Oishi K, & Wullaert A. (2016). Bifidobacteria Prevent Tunicamycin-Induced Endoplasmic Reticulum Stress and Subsequent Barrier Disruption in Human Intestinal Epithelial Caco-2 Monolayers. PLoS ONE, 11(9), e0162448.

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Fuziline Modulates Mitochondrial Dysfunction

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Fuziline (CHB171026, MF: C24H39O7N, MW: 453.58, purity: 99.3%) was obtained from Chengdu Chroma‐Biotechnology Co., Ltd. (China) and prepared by dissolving in dimethyl sulfoxide as a 50 mmol/L stock solution. 3‐(4, 5‐dimetrylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) and ISO were purchased from Sigma‐Aldrich LLC (USA). 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanine iodide (JC‐1), 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) and MitoSOX were obtained from Beyotime Biotechnology Co., Ltd. (China). Annexin‐V‐FLUOS Staining Kit and Tunel Kit were purchased from Roche Co., Ltd. (USA). N‐acetylcysteine (NAC) and GSK2606414 were purchased from MedChemExpress Co., Ltd. (China). Antibodies against caspase‐3, p‐PERK, PERK, p‐eIF2α, eIF2α, GRP78, Chop, ATF4 and cytochrome C were obtained from Cell Signaling Technology Co., Ltd. (USA). Bcl‐2, Bax and GAPDH were purchased from Servicebio (China).

Fan C., Yao Z., Ye M., Fu L., Zhu G., Dai Y, & Yao X. (2019). Fuziline alleviates isoproterenol‐induced myocardial injury by inhibiting ROS‐triggered endoplasmic reticulum stress via PERK/eIF2α/ATF4/Chop pathway. Journal of Cellular and Molecular Medicine, 24(2), 1332-1344.

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Comprehensive Western Blotting Workflow

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The cell lysing, western blotting and immunoprecipitation were performed as previously described^{29 (link)}. The following antibodies were used: OGT, OGA, GAPDH, P-gp, AKT (Rabbit), XBP1, GFAT, PFK and G6PD were from Abcam (Hong Kong, China). pAKT (S473), AKT (mouse), GRP78, Caspase-9 and Caspase-3 were from Cell Signaling Technology (MA, USA); O-GlcNAcylation antibody (CTD110.6) was from BioLegend (MA, USA). Chemiluminescent detection was performed using ECL kit (GE Healthcare, USA).

Liu Y., Cao Y., Pan X., Shi M., Wu Q., Huang T., Jiang H., Li W, & Zhang J. (2018). O-GlcNAc elevation through activation of the hexosamine biosynthetic pathway enhances cancer cell chemoresistance. Cell Death & Disease, 9(5), 485.

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Protein Quantification and Antibody Validation

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The protein level was determined, according to the method of Tan et al. [31 (link)]. The antibodies used in this study included: PPARα (Absin, Shanghai, China), CPT1 (Proteintech, USA), FXR (Bioss, Beijing, China), HNF1α (Abcam, Cambridge, UK), GRP78 (Cell Signaling Technology, CST, Danvers, MA, USA), p-PERK (Bioss, Beijing, China), p-EIF2α (CST, Danvers, MA, USA), JNK (CST, Danvers, MA, USA), p-JNK (CST, Danvers, MA, USA), P38 (CST, Danvers, MA, USA), p-P38 (CST, Danvers, MA, USA), ERK(CST, Danvers, MA, USA), p-ERK(CST, Danvers, MA, USA), c-Jun (CST, Danvers, MA, USA), p-c-Jun (CST, Danvers, MA, USA), GAPDH (ZSGB-Bio, Beijing, China), FLAG (CST, Danvers, MA, USA), HA (CST, Danvers, MA, USA) and GFP (CST, Danvers, MA, USA). An electrochemiluminescence kit (Beyotime, Beijing, China) was used to visualize the immunoreactive protein, which was then scanned with an Epson Perfection V33 scanner.

Du J., Xiang X., Xu D., Zhang J., Fang W., Xu W., Mai K, & Ai Q. (2021). FXR, a Key Regulator of Lipid Metabolism, Is Inhibited by ER Stress-Mediated Activation of JNK and p38 MAPK in Large Yellow Croakers (Larimichthys crocea) Fed High Fat Diets. Nutrients, 13(12), 4343.

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Comprehensive Signaling Pathways Analysis

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Antibodies to BAX, BAK, BCL-XL, CHOP, c-FLIP, IRE1, RIP1, iNOS, FADD, Cathepisin B, mTOR, phospho-mTOR S2448 and S2481, phospho-RAPTOR S792, TSC2 T1426, PTEN, phospho-PTEN S380, ATF6, eNOS, AIF, XBP1, NOXA, PUMA, ATG5 phospho-ATG13 S318, Beclin-1, AKT, phospho-AKT T308, eiF2α, phospho-eiF2α S51, phospho p65 S536, ATF4, PGKI and II, TRX, SOD, ATM, phospho-ATM S1981, AMPKα, phospho-AMPK T172, phospho-ULK1 S757, S317, STAT3, STAT5, p70 S6K, phospho-ERK1/2, GRP78, HSP70 and HSP90, phospho-γH2AX, were purchased from Cell Signaling Technology, (Danvers, MA). PERK, CD95 and caspase 9 antibodies, were purchased from Santa Cruz Biotechnology, (Dallas, TX).

Roberts J.L., Poklepovic A, & Booth L. (2017). Curcumin interacts with sildenafil to kill GI tumor cells via endoplasmic reticulum stress and reactive oxygen/ nitrogen species. Oncotarget, 8(59), 99451-99469.

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Western Blot Analysis of ER Stress Markers

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Preparation procedures and characterization data for C-6 and its analogs can be found in a supplementary file (Additional file 1). The following primary antibodies were purchased from Cell Signaling (Danvers, MA, USA): CHOP, GRP78, p-EIF2α, pan-EIF2α, p-JNK, and pan-JNK. In addition, the vinculin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Actinomycin D (ACTD) and cycloheximide (CHX) were also obtained from Sigma-Aldrich.

Vaden R.M., Gligorich K.M., Jana R., Sigman M.S, & Welm B.E. (2014). The small molecule C-6 is selectively cytotoxic against breast cancer cells and its biological action is characterized by mitochondrial defects and endoplasmic reticulum stress. Breast Cancer Research : BCR, 16, 472.

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Western Blot Analysis of Protein Expression

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Protein was extracted from the lysed VCMs as previously described [18 (link)]. The total protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, 23225). Equal amounts of protein were separated by electrophoresis on SDS-PAGE gels (Beyotime, P0012A) and then blotted onto nitrocellulose membranes. Then, the membranes were incubated with primary antibodies overnight. The following primary antibodies were used for the western blotting: GRP78 (1:1000, Cell Signaling, Danvers, MA, USA, 3177), CHOP (1:1000, Cell Signaling, 2895), calpain (1:1000, Cell Signaling, 2539), cTnT (1:1000, Cell Signaling, 5593), caspase-3 (1:1000, Cell Signaling, 9662), Bcl-2 (1:1000, Cell Signaling, 3498), Bax (1:1000, Cell Signaling, 2772), L-type calcium channel (1:1000, Abcam, ab96713), β-tubulin (1:1000, Cell Signaling, 2148) and GAPDH (1:1000, Cell Signaling, 2118S). Secondary antibodies used were anti-rabbit IgG, HRP-linked antibody (1:5000, Cell Signaling, 7074P2) and anti-mouse IgG, HRP-linked antibody (1:5000, Cell Signaling, 7076). After the addition of the chemiluminescent reagents (Thermo Fisher Scientific, 17295), the bands were detected using a Molecular Imager ChemiDoc™ XRS+ Imaging System (Bio-Rad) and quantified using the Image Lab™ Software (Bio-Rad).

Cui C., Geng L., Shi J., Zhu Y., Yang G., Wang Z., Wang J, & Chen M. (2017). Structural and electrophysiological dysfunctions due to increased endoplasmic reticulum stress in a long-term pacing model using human induced pluripotent stem cell-derived ventricular cardiomyocytes. Stem Cell Research & Therapy, 8, 109.

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Vitamin D and ER Stress Response

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The 1,25-dihydroxyvitamin D₃ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM)/F12, and trypsin were purchased from Hyclone (Logan, UT, USA). The CCK-8 assay was purchased from Dojindo (Japan). Annexin V-FITC/PI was purchased from Yeasen Corporation (Shanghai, China). A PrimeScript RT Reagent Kit and SYBR Premix Ex Taq™ II were purchased from TaKaRa (Tokyo, Japan). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Primers targeting glucose-regulated protein (GRP) 78, ATF4, CHOP, VDR, and GAPDH were synthesized by Sangon Biotech (Shanghai, China). Radio immunoprecipitation assay (RIPA) lysis buffer, a bicinchoninic acid (BCA) protein assay kit, antibody solution, and 5x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) same loading buffer were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies (VDR, PERK P-PERK, eIF-2α, P-eIF-2α, ATF4, CHOP, and GRP78) were purchased from Cell Signaling Technology (Beverly, MA, USA). Polyvinylidene difluoride membranes (0.45 mm) were purchased from Millipore (Billerica, MA, USA). Human serum albumin (HSA; 1.5 mmol/l) was purchased from Sigma-Aldrich.

Yuan Y.F., Das S.K, & Li M.Q. (2018). Vitamin D Ameliorates Impaired Wound Healing in Streptozotocin-Induced Diabetic Mice by Suppressing Endoplasmic Reticulum Stress. Journal of Diabetes Research, 2018, 1757925.

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