Harvested cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA, USA) for 20 minutes and centrifuged at 13,500 rpm for 5 minutes. Twenty micrograms of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 8%–12% gels and transferred to nitrocellulose membranes (Potran nitrocellulose membrane, Whatman, Kent, UK) using an iBlot dry blotting system (Invitrogen). The membranes were blocked with 5% non-fat dried milk and incubated with specific primary antibodies for (1) GADD34 (1:1,000), activating transcription factor 4 (ATF4; 1:1 000), p53 (1:1,000), and β-actin (1:2,000) from Santa Cruz Biotechnology (Dallas, TX, USA); (2) eIF2 (1:1,000), phospho-eIF2 (p-eIF2, 1:1,000), p-p53 (Ser15, 1:2,000), p21Waf1/Cip1 (1:500), cyclin B1 (1:1,000), cyclin E2 (1:1,000), p-cdc2 (p-cdk1, 1:2,000), Bax (1:1,000), Bcl-xL (1:2,000), procaspase-9 (1:2,000), and cleaved caspase-9 (1:1,000) from Cell Signaling Technology. After incubation with appropriate horseradish peroxidase-conjugated secondary IgG antibodies, bands were detected using ECL reagent (Amersham-GE Healthcare Life Sciences, Pittsburgh, PA, USA).
Gadd34
Manufactured by Santa Cruz Biotechnology
Sourced in United States
GADD34 is a protein phosphatase regulatory subunit that is involved in the cellular stress response. It functions to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), which can lead to a reduction in global protein synthesis. GADD34 is an important component of the integrated stress response pathway.
Automatically generated - may contain errors
Lab products found in correlation
Eif2α, by Cell Signaling Technology (6 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
P perk, by Santa Cruz Biotechnology (3 mentions)
P eif2α, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
P21 waf1 cip1, by Cell Signaling Technology (1 mentions)
Procaspase 9, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Cyclin e2, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Ab6046, by Abcam (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
Ppp1r15b, by Proteintech (1 mentions)
Aprotinin, by Merck Group (1 mentions)
9 protocols using gadd34
1
Western Blot Analysis of Cellular Signaling
2
Western Blot Analysis of Patient-Derived Cells
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Western blot analysis was conducted to assess protein levels in patient-derived lymphoblast cells. Cells were lysed in radioimmunoprecipitation assay buffer containing 10 mg/ml each of aprotinin, phenylmethanesulfonyl fluoride and leupeptin (all from Sigma) for 20 min at 4°C, followed by centrifugation at 13 000×g for 15 min and retrieval of supernatants. Total protein concentrations were determined by Bradford protein assay (BioRad Laboratories). Protein samples were resolved by 11% SDS–PAGE, transferred onto nitrocellulose membrane and incubated in blocking solution (Tris-buffered saline (TBS), 5% non-fat milk, 0.05% Tween-20) for 1 h at room temperature followed by overnight incubation with primary antibody at 4°C (PPP1R15B, Proteintech 14634-1-AP; GADD34, Santa Cruz Biotechnology, Inc. sc-8327; GAPDH, ImmunoChemical, Inc. 200-901-BJ4; β-Tubulin and PPP1c, Abcam ab6046 and ab16387; p-eIF2α and total eIF2α, Cell Signalling 9721 and 9722). Membranes were washed with TBS and 0.5% Tween-20 three times followed by incubation with secondary antibody (HRP-conjugated anti-rabbit or anti-mouse; Cell Signalling) for 1 h at room temperature. Blots were visualized by autoradiography using the Clarity Western ECL substrate (BioRad Laboratories).
3
Endoplasmic Reticulum Stress Signaling
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RPMI 1640 was obtained from Thermo-Fisher Biochemical Products Co. Ltd (Beijing, China). Fetal bovine serum (FBS), thapsigargin (TG), protein kinase A (PKA) inhibitor H89 and KT5720, 4-phenylbutyric acid (PBA) and PGE1 were purchased from Sigma (St. Louis, MO, United States). Antibodies against GRP78, PERK, eukaryotic translation initiation factor-2α (eIF-2α), phospho-PERK (p-PERK) and phospho-eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), spliced X box-binding protein 1 (sXBP1), growth arrest and DNA damage-inducible gene 34 (GADD34) and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was purchased from Dojindo Laboratories (Kumamoto, Japan). Small interfering (si)RNA scramble control and validated human GRP78-siRNA were purchased from Santa Cruz Biotechnology. All other chemicals and reagents were obtained from Sigma, unless stated otherwise.
4
Optimizing Angiogenic Signaling Pathways
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Recombinant human BMP-2, anti–α-tubulin antibody, anti–β-actin antibody, collagenase IA-S, protease inhibitor cocktail, and phosphatase inhibitor cocktails I and II were purchased from Sigma-Aldrich. Recombinant human TNF, GM-CSF, VEGF165, PDGF-BB, and mouse IgG were purchased from R&D Systems. BMPR2 and Mac-3 antibodies were purchased from BD, and S-6, L13a, p38, p-p38, MK2, p-MK2, I-κB, p–I-κB, eIF2α, and p-eIF2α antibodies were purchased from Cell Signaling Technology. GADD34, protein phosphatase-1, GM-CSFRα, HuR, and TIA-1 antibodies (4H1) and salubrinal were obtained from Santa Cruz Biotechnology, Inc., and G3BP antibody was obtained from BD. SB202190 was purchased from EMD Millipore. GM-CSF and TNF antibodies for immunohistochemistry were obtained from Abcam, and CD31, CD34, CD68, and α-SM actin antibodies and normal rabbit Ig were obtained from Dako. HRP-conjugated rabbit and mouse secondary antibodies and ECL and ECL Plus kits were ordered from GE Healthcare. Allophycocyanin (APC)-human CD31 and PE–GM-CSFRα antibodies were purchased from eBioscience. Recombinant murine GM-CSF was obtained from PeproTech.
5
Cell Lysis and Immunoblot Analysis
6
Western Blot Analysis of UPR Markers
7
Huh-7.5 Cell Infection with HCV
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Huh-7.5 cells were obtained from Charles M. Rice (Rockefeller University, New York). The Huh-7.5 cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, sodium pyruvate, nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Huh-7.5 cells were infected with JFH1-GFP chimera HCV using a protocol developed in our laboratory as previously described31 (link). Recombinant human IFN-α was purchased from Schering Plough, Kenilworth, NJ. Sofosbuvir was obtained from Acme Biosciences, Inc. (Palo Alto, CA) and ledipasvir was obtained from Selleck Chemicals (Houston, TX). Thapsigargin (TG), tauroursodeoxycholic acid (TUDCA), PERK inhibitor (GSK 2606414), α-tocopherol, and roscovitine were obtained from Sigma-Aldrich (St. Louis, CA). Antibody to pNrf2 was purchased from Abcam. Antibodies to GRP78 (BiP), PERK, eIF2α, peIF2α, CHOP, IRE1, GFP and Rb were obtained from Cell Signaling (Beverly, MA). Antibody to NS3 was purchased from Virogen Inc. Antibodies to HCV core and pIRE1 were purchased from Thermo Scientific. Antibody to GRP94, GADD34, pPERK, ATF4, XBP1, ATF6, Mdm2, β-actin and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
8
Western Blot Analysis of ER Stress Markers
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Whole protein extracts were recovered from cells after treatment with DMSO or SIX2G by using NP40 lysis buffer complemented with Halt Protease and Phosphatase Inhibitor cocktail (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA). Western blot (WB) analysis was performed as previously reported [26 (link)]. The primary antibodies used were: PERK (#3192), eIF2α (#5324), p-eIF2α (Ser 51) (#3398), Caspase-3 (#9662), Cleaved Caspase-3 (Asp 175) (#9661), Caspase-8 (#4790) and Cleaved Caspase-8 (#9748) by Cell Signaling Technology (Danvers, MA, USA); p-PERK (Thr 981) (#orb336657), b-actin (#Sc-1616), GAPDH (SC-25778), GADD34 (SC-46661) and PP1 (SC-7482) by Santa-Cruz (Dallas, TX, USA). Anti-rabbit IgG HRP-linked antibody (#7074, Cell Signaling Technology, Danvers, MA, USA), anti-mouse IgG HRP-linked antibody (#7076, Cell Signaling Technology, Danvers, MA, USA) and anti-goat IgG HRP-linked antibody (SC-2354, Santa Cruz, Dallas, TX, USA) were used as secondary antibodies depending on the host species of animal in which the primary antibodies were raised.
9
Immunoblotting for Protein Analysis
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Immunoblotting experiments were conducted as previously described [31 (link)]. Antibodies used for immunoblotting were specific for the proteins as follows: BiP, P-eIF2α, eIF2α, PARP, Synoviolin1, PERK, and IRE1α (Cell Signaling); GADD34, P-PERK, XBP1, ATF6, MDM2, and p53 (Santa Cruz); and β-actin (Sigma). Antibodies were diluted to 1:1000, except for anti-β-actin (1:10000). Secondary antibodies were purchased from Promega (antirabbit and antimouse at 1:5000) or Rockland (TrueBlot antimouse at 1:1000).
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