Cells were harvested and detergent-solubilized cell lysates were obtained as described previously [5 (link), 7 (link), 8 (link), 17 (link)]. Equal amounts of cell lysates were resolved by SDS-PAGE and subjected to western blot analysis. To examine the interaction between eIF2α and PHLPP, the solubilized cell lysates were incubated with either the anti-HA high-affinity antibody or the anti-PHLPP1 or PHLPP2 antibodies and protein A/G agarose beads (Thermo) at 4 °C for overnight. The beads were washed three times with lysis buffer and the immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting. The phospho-eIF2α, total eIF2α, ATF4, and LC3 antibodies were obtained from Cell Signaling. The PHLPP1 and PHLPP2 antibodies were from Bethyl laboratories. The β-actin and the anti-HA high-affinity antibodies were from MilliporeSigma.
Phospho eif2α
Manufactured by Cell Signaling Technology
Sourced in United States, China
Phospho-eIF2α is a laboratory reagent that detects the phosphorylated form of the eukaryotic translation initiation factor 2 alpha (eIF2α) protein. Phosphorylation of eIF2α is a key regulatory event in the control of protein synthesis in response to various cellular stresses.
Automatically generated - may contain errors
Lab products found in correlation
Eif2α, by Cell Signaling Technology (48 mentions)
P erk, by Cell Signaling Technology (21 mentions)
Cleaved caspase 3, by Cell Signaling Technology (17 mentions)
β actin, by Merck Group (15 mentions)
Total eif2α, by Cell Signaling Technology (11 mentions)
β actin, by Cell Signaling Technology (11 mentions)
Phospho perk, by Cell Signaling Technology (10 mentions)
Ire1α, by Cell Signaling Technology (10 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Caspase 3, by Cell Signaling Technology (8 mentions)
Phospho jnk, by Cell Signaling Technology (8 mentions)
Phospho akt, by Cell Signaling Technology (7 mentions)
Phospho erk1 2, by Cell Signaling Technology (6 mentions)
Phospho p38, by Cell Signaling Technology (6 mentions)
Eif2α, by Santa Cruz Biotechnology (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Erk1 2, by Cell Signaling Technology (5 mentions)
Bcl 2, by Cell Signaling Technology (5 mentions)
Beclin 1, by Cell Signaling Technology (5 mentions)
Cleaved parp, by Cell Signaling Technology (4 mentions)
105 protocols using phospho eif2α
1
Elucidating eIF2α-PHLPP Interaction
2
Autophagy Modulation in Cell Survival
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PT (purity ≥ 98%) and 3-methyladenine (3-MA) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Chloroquine (CQ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 4-phenylbutyric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for p62 and LC3 were purchased from Novus Biologicals (Littleton, CO, USA), and antibodies for cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerase (PARP), Bip, PERK, eIF2α, phospho-eIF2α, ATF4, calreticulin and CHOP (C/EBP homologous protein) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for Beclin-1, lamin B, α-tubulin, salubrinal (Sal), E-64d and pepstatin A (lysosomal protease inhibitors), small interfering RNA (siRNA)-eIF2α (si-eIF2α) and siRNA-LC3 (si-LC3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3
Gambogic Acid and Mitochondrial Dysfunction
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Gambogic acid (GA) was purchased from Biorbyt (San Francisco, CA, UK). Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) was from Calbiochem (EMD Millipore Corp., Billerica, MA, USA). MitoTracker-Red (MTR), propidium iodide (PI), 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2DCF-DA), and tetramethylrhodamine methyl ester (TMRM) along with secondary antibodies (rabbit IgG HRP (G-21234) and mouse IgG HRP (G-21040)) were from Molecular Probes (Eugene, OR, USA). The primary antibody against SDHA (ab14715) was from Abcam (Cambridge, MA, USA) and Noxa (OP180) was from Calbiochem. β-actin (sc-47778), ubiquitin (sc-8017), Mcl-1 (sc-819), ATF4 (sc-200) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SAPK/JNK (#9251), SAPK/JNK (#9252), phospho-ERK1/2 (#9101), ERK1/2 (#9102), TCF11/NRF1 (#8052), CHOP/GADD153 (#2895), phospho-eIF2α (#9721), and eIF2α (#9722) were from Cell Signaling Technology (Danvers, MA, USA). PDI (ADI-SPA-890-F) was form Enzo Life Science (Farmingdale, NY, USA). Other reagents were from Sigma-Aldrich (St Louis, MO, USA).
4
Protein Extraction and Western Blot Analysis
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Cells were lysed in buffer containing the following: 50 mmol/L TrisHCl pH 6.7, 150 mmol/L NaCl, 1% Triton X-100, cOmplete Protease Inhibitor Cocktail (Roche), Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich), and Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich). The following antibodies were used: anti-Wfs1 (11558–1-AP; Proteintech), phospho-PERK (3179; Cell Signaling), phospho-Eif2α (3398; Cell Signaling), total Eif2α (9722S; Cell Signaling), total PERK (3192S; Cell Signaling), GAPDH (G8795; Sigma-Aldrich), Spry2 (ab50317; Abcam), and Serca2 (sc-8095; Santa Cruz Biotechnology). Islet protein samples were isolated from the TRIzol Reagent organic phase after RNA isolation as described in the protocol, except the pellet was resuspended in 5 mol/L urea in 0.5% SDS.
5
Antibody Detection for Cellular Signaling
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Antibodies (Abs) against MAP1LC3B, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, Smad1, phospho-Ser206-Smad1, GSK3β, phospho-Ser9-GSK3β, active caspase 7, active caspase 9, phospho-eIF2α and eukaryotic translation initiation factor 2 alpha (eIF2α) were purchased from Cell Signaling Technology, Inc. Danvers, MA, USA. Anti-Gap43 and β-actin Abs were purchased from Santa Cruz Biotechnology, Inc. Heidelberg, Germany. Anti-mitochondrial Cytochrome C Oxidase I (mtCO1), VDAC1, Tubulin β-III, MAP2 and Aconitase1 (ACO1) Abs were purchased from Abcam, Cambridge, UK. Anti-DDK Ab was purchased from OriGene Technologies. Anti-OPA1 and Drp1 Abs were purchased from Becton, Dickinson and Company, Franklin Lakes, NJ, USA. Anti-p62 and BNIP3 Abs were purchased from Sigma Aldrich.
6
Dissecting Apoptosis Signaling Pathways
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G1 was purchased from Tocris (Wiesbaden-Nordenstadt, Germany), dissolved (5 mM) in dimethyl sulfoxide (DMSO) (Roth, Karsruhe, Germany) and stored at −20 °C; Thapsigargin, SP600125, GSK2606414 were also purchased from Tocris. Indo-1 AM was from Thermo Fisher Scientific (Waltham, MA, USA). zVAD-fmk was bought from Santa Cruz (Santa Cruz, CA, USA). SB203580 and Kira6 were purchased from MERCK Millipore (Darmstadt, Germany). All substances were dissolved in DMSO. Antibodies were obtained from the following commercial sources: caspase 9 (Ca# 9502), cleaved PARP (Ca# 9541), IRE1α (Cat# 3294), PERK (Cat# 3192), eIF2α (Cat# 5234), phospho-eIF2α (Cat# 3398), BiP GRP78 (Ca# 3177), CHOP (Cat# 2895), p38 MAPK (Ca# 9212), phospho-p38 MAPK (Ca# 4511), phospho-SAPK/JNK (Ca# 4668), caspase 3 (Ca# 9662), BCL-2 (Ca# 2872), Cell Signaling (Danvers, MA, USA); ATF6 (Cat# 73500), BioAcademia (Osaka, Japan); puromycin (Ca# MABE343), cylophilin D (Ca# AP1035), MERCK Millipore (Darmstadt, Germany); phospho-IRE1α (Cat# NBP2-50067), Novus Biologicals (Littleton, CO, USA); cytochrome c (Ca# 556433), BD Biosciences (Franklin Lakes, NJ, USA); β-actin (Cat# A5441, Sigma-Aldrich (Steinheim, Germany)). Secondary, peroxidase-conjugated antibodies were purchased from Dianova (Hamburg, Germany). All other chemicals of analytical grade were obtained from Sigma-Aldrich or Roth.
7
Cellular Stress Response Protocol
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), paclitaxel (PTX), bafilomycin A1 (BAF-A1), N-acetyl-L-cysteine (NAC), cycloheximide (CHX), catalase-polyethylene glycol (PEG), 4,6-diamidino-2-phenylindole (DAPI), and reduced glutathione (GSH) were purchased from Sigma-Aldrich. 3-Methyladenine (3-MA) and Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were purchased from Merck Millipore. ERK/MEK1 inhibitor (selumetinib) was purchased from Selleck Chemicals. DMEM, FBS, and the antibiotics mixture (penicillin-streptomycin) were purchased from Invitrogen. Primary antibodies against β-actin (Chemicon, Millipore), p62 (BD Biosciences), LC3A/B and Ki-67 (Abcam), LC3B (Arigo Biolaboratories.), Raf1 (GeneTex), PDI, caspase-7, Bim, phospho-eIF2α, phospho-ERK1/2, phospho-SAPK/JNK, phospho-p38 MAPK (Cell Signaling Technology) were used. All other antibodies were obtained from Santa Cruz Biotechnology.
8
Nelfinavir Mesylate Purification Protocol
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Nelfinavir mesylate (NFR) powder was extracted and purified from 250 mg tablets (Agouron pharmaceuticals; San Diego, CA). Curcumin (CUR) was obtained from Acros Organics (Fair Lawn, NJ). Docetaxel (DTX) and Thapsigargin (Tg) were obtained from Sigma (St. Louis, MO) and the AKT-inhibitor (AKTi-IV; Catalog no. 124005) was obtained from Calbiochem (Billerica, MA). For in vitro studies, all drugs were dissolved in dimethyl sulfoxide (DMSO). Primary antibodies against total-AKT and phospho-AKT, total eIF2α and phospho-eIF2α, and against human BiP/Grp78, PARP and CHOP, were all purchased from Cell Signaling Technology (Danvers, MA). Antibodies against human TRIB3 and ATF4 were from Santa Cruz Biotechnology (Santa Cruz, CA), antibody against human β-Actin was from Fisher Scientific (Waltham, MA) and against Ki-67 was from Spring Biosciences (Pleasanton, CA). All secondary antibodies, such as goat anti-mouse, goat anti-rabbit and bovine anti-goat, were all purchased from Santa-Cruz Biotechnology.
9
Investigating Cellular Stress Pathways
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3-TYP (S8628) was obtained from MedChemExpress (China). TAA 163678) were obtained from Sigma-Aldrich (United States). Phospho-eIF2α (#3398), eIF2α (#5324), CHOP(#2895), phospho-JNK (#4668), JNK (#9252), phospho-ERK1/2 (#4370), ERK1/2 (#9102), phospho-P38 (#4511), P38 (#8690), phospho-NF-κB-p65 (#3033), NF-κB-p65 (#8242), Nrf2 (#12721), Lamin B1 (#13435), HO-1 (#43966), Cleaved caspase 3 (#9664), TNF-α (#11948), IL-1β (#12426), SIRT1 (#8469), SIRT2 (#12672), SIRT3 (#5490), MnSOD (#13194), and GAPDH (# 5174) were obtained from Cell Signaling Technology (CST) (United States), ALDH2 (15310-1-AP) antibody was obtained from Proteintech (China).
10
Protein Extraction and Western Blot Analysis
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Cells were lysed on ice in CelLytic MT Cell Lysis Reagent (Sigma) supplemented with protease and phosphatase inhibitors. Protein concentration was determined with Pierce BCA Protein Assay Kit (Thermo Fisher). Protein lysates were resolved on SDS-PAGE gels and transferred to PVDF membranes [36 (link)]. The following antibodies were used for the western blot analysis: phospho-eIF2α (#9721, 1:1000), eIF2α (#9722, 1:1000), ATF4 (#11815, 1:1000), BIM (#2819, 1:1000), SYVN1 (#14773, 1:1000) from Cell Signaling Technology; phospho-PERK (Thr980) (MA5–15033, 1:1000, Thermo Fisher); SEL1L (S3699, 1:1000, Sigma); β-tubulin (10094–1-AP, 1:5000, Proteintech).
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