Liver tissues were homogenized and sonicated in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitor cocktail and phosphostop (Roche Diagnostics, Indianapolis, IN), and total protein was extracted by centrifugation. A total of 30 μg protein was resolved on 4–12% precast SDS-PAGE gel (Invitrogen, Grand Island, NY) and transblotted onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). Membranes were probed with antisera against p-JNK, JNK, p-PDK, PDK, p-AKT, AKT, p-PKCζ, PKCζ, p-PERK, PERK, p-elF2α, elF2α, p-IRE-1, IRE-1, GRP78, CHOP, caspase-3, cleaved caspase-3, ATF-6α, XBP-1, BCL-2, Bax, LC-3B and β-actin (Cell Signaling Technology, Danvers, MA). Isotype-matched horseradish peroxidase conjugated secondary antibodies, enhanced chemiluminescence substrate (Pierce, Rockford, IL) and a FluorChem 8900 digital imaging system (AlphaInnotech, San Leandro, CA) were used to visualize protein bands. Densitometric analyses was performed with VisionWorks® Software, version 6.8 (UVP, Upland, CA).
Elf2α
Manufactured by Cell Signaling Technology
Sourced in United States
The ElF2α is a lab equipment product that plays a core role in the regulation of protein synthesis. It is a key component of the eukaryotic translation initiation complex, which is essential for the initiation of protein translation. The ElF2α specifically binds to GTP and initiator methionyl-tRNA, facilitating their recruitment to the 40S ribosomal subunit.
Automatically generated - may contain errors
Lab products found in correlation
P elf2α, by Cell Signaling Technology (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Grp78, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Parp1, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail and phosphostop, by Roche (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Precast sds page gel, by Thermo Fisher Scientific (2 mentions)
P ire1, by Cell Signaling Technology (2 mentions)
Atf6α, by Cell Signaling Technology (2 mentions)
Fluorchem 8900 digital imaging system, by Bio-Techne (2 mentions)
Visionworks software version 6, by Analytik Jena (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Phospho elf2α, by Cell Signaling Technology (2 mentions)
Cytochrome c, by Cell Signaling Technology (2 mentions)
Rabbit anti human β actin antibody, by Merck Group (2 mentions)
P perk, by Cell Signaling Technology (2 mentions)
10 protocols using elf2α
1
Western Blot Analysis of Liver Proteins
2
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were prepared either in 20 mM Tris pH 7.9, 120 mM KCl, 5 mM MgCl2 0.2% Nonidet P-40, 5 mM EDTA, 10% glycerol (for PARP1 and Casp3 analysis) or in 100 mM Tris pH 8, 140 mM NaCl, 20 mM EDTA, 0.2% SDS, 1% Nonidet P-40 lysis buffer, resolved on SDS-PAGE gels and transferred onto PVDF membranes using the iBlot dry Transfer System (Life Technologies, Carlsbad, CA, USA). Blocked membranes were incubated with the following primary antibodies: Atf-4 (1 : 500); elF2α (1 : 1000); phospho-elF2α (1 : 500); PARP1 (1 : 1000, Cell Signaling Technology, CST, Danvers, MA, USA), which detects the full-length protein (116 kDa) as well as the large caspase-cleaved fragment (89 kDa); anti-Casp3 (1 : 1000) all from CST, and anti-LC3 (2 μg/ml, Novus Biologicals, Littleton, CO, USA) anti-Gapdh (1 : 10 000, Abcam, Cambridge, UK), followed by the appropriate HRP-conjugated secondary antibodies (1 : 10000, Dako, Glostrup, Denmark). Detection was performed with ECL reagents (Pierce, Thermo Scientific, Waltham, MA, USA). The ImageJ software (open source; http://imagej.nih.gov/ij ) was used for densitometric quantification.
3
Protein Expression Analysis of Flaccidoxide-13-acetate
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Rabbit anti-human ERK, p-ERK, JNK, p-JNK, GRP78, ATF4, and cleaved-ATF6 antibodies were purchased from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against AKT, p-AKT, PI3K, p-PI3K, Mcl-1, Bad, p-Bad, Bcl-xl, Bcl-2, Bax, p38, p-p38, PERK, p-PERK, elF2α, p-elF2α, pro-caspase 3, cleaved caspase 3, pro-caspase 9, cleaved caspase 9, cytochrome C, and CHOP were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Cytosolic cytochrome C were separated using a cytochrome C releasing apoptosis assay kit (Biovision, Milpitas, CA, USA).
The flaccidoxide-13-acetate treated sample and DMSO treated control samples (total protein 25 μg) were separated by 12.5% SDS-PAGE, and the proteins on the gel were transferred to a PVDF membrane. The membrane containing transferred protein was blocked in PBS buffer and incubated with primary antibody at 4 °C overnight, followed by secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk) for 2 h at 4 °C. The signals were detected with an enhanced chemiluminescence detection kit.
The flaccidoxide-13-acetate treated sample and DMSO treated control samples (total protein 25 μg) were separated by 12.5% SDS-PAGE, and the proteins on the gel were transferred to a PVDF membrane. The membrane containing transferred protein was blocked in PBS buffer and incubated with primary antibody at 4 °C overnight, followed by secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk) for 2 h at 4 °C. The signals were detected with an enhanced chemiluminescence detection kit.
4
Protein Expression and Activation Analysis
5
Quantitative Western Blot Analysis
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Protein samples were prepared from liver homogenates in Laemmli sample buffer, run on SDS-polyacrylamide gels (4-15% TGX stain-free gel, Bio-Rad), and transferred to the polyvinylidene difluoride (PVDF) membrane. The membranes were blocked, incubated with primary antibodies overnight at 4 °C, followed by secondary antibodies, and developed using the chemiluminescence imaging system (Bio-Rad). Following primary antibodies were used: OPA1 (BD Biosciences, 612606; 1:1000); caspase-3 (Cell Signaling, 9662; 1:1000), PARP-1 (Cell Signaling, 9542; 1:1000), β-actin (Sigma, A1978; 1:40000), TOM20 (Proteintech, 11802-1-AP; 1:1000), cytochrome c (BD Biosciences, 556432; 1:5000), elF2α (Cell Signaling, 9722; 1:1000), phospho-elF2α (Cell Signaling, 9721; 1:1000), FGF21 (Proteintech, 26272-1-AP; 1:1000), LC3 A/B (Cell Signaling, 4108; 1:1000), PGC1α (Invitrogen, PA5-38022; 1:500), OMA1 (Santa Cruz, sc-515788; 1:100), and mitochondria total OXPHOS rodent WB cocktail (Abcam, ab110413; 1:1000). JNK (Cell Signaling, 9252; 1:1000), p-JNK-Thr183/Tyr185 (Cell Signaling, 9255; 1:500), MCU (Sigma, HPA016480; 1:1000), NCLX (Proteintech, 21430-1-AP; 1:1000), CypD (Proteintech, 18466-1-AP; 1:1000), MnSOD (BD Biosciences, 611580; 1:1000), GPx1/2 (Santa Cruz Biotechnology, sc-133160; 1:200), and CYP2E1 (Proteintech, 19937-1-AP; 1:300).
6
Renal Cancer Cell Lines Characterization
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The renal cancer cell 786-O (BCRC. 60243), A-498 (BCRC. 60241) and immortalized normal proximal tubule epithelial cells (HK-2, BCRC. 60097) were purchased from Bioresources Collection and Research Center (BCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). ACHN and CaKi-1 cells were kindly donated by Dr. M.H. Chien (Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan). 786-O cells were maintained in RPMI-1640 (Gibco, Thermo Fisher Scientific, USA), and CaKi-1, A-498, and ACHN cells were maintained in MEM (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% FBS (Hyclone, USA), 10 mM of glutamic acid, and 1% penicillin/streptomycin. All cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.
NCTD, MTT, and JC-1 were purchased from Sigma (St. Louis, MO, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from BioVision (Mountain View, CA). Western blotting antibodies against p-p38, p-JNK, p-AKT, ERK1/2, p38, JNK1/2, AKT, Mcl-1, Bcl-2, Bax, and β-actin were purchased from Santa Cruz Biotechnology (California, USA). Antibodies against HA, p-ERK1/2, p-elF2α, elF2α, cleaved-caspase-3, -caspase-6, -caspase-7, -caspase-8, -caspase-9, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA).
NCTD, MTT, and JC-1 were purchased from Sigma (St. Louis, MO, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from BioVision (Mountain View, CA). Western blotting antibodies against p-p38, p-JNK, p-AKT, ERK1/2, p38, JNK1/2, AKT, Mcl-1, Bcl-2, Bax, and β-actin were purchased from Santa Cruz Biotechnology (California, USA). Antibodies against HA, p-ERK1/2, p-elF2α, elF2α, cleaved-caspase-3, -caspase-6, -caspase-7, -caspase-8, -caspase-9, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA).
7
Antibody-based Western Blot Analysis
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Antibodies used for Western blot were purchased from Cell signaling technology, Danvers, MA, USA: AKT (pan) (C67E7), phospho-S473-AKT (D9E), phosphor-T308 AKT (244F9), AMPKα, phospho-T172-AMPKα (40H9), p70-S6K, phospho-T389-p70-S6K, S6P (5G10), phospho-S235/236-S6P, Raptor (24C12), Rictor (53A2), elF2α, phospho-S51-elF2α (119A11), ULK1 (D8H5), phospho-S757-ULK1, phospho-S555-ULK1 (D1H4), 4-EBP1 and LC3B (D11) XP. Antibodies used for immunocytochemistry: elF3η (C-5) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA, and G3BP1 was purchased from Thermo Fischer Scientific, Waltham, MA, USA.
8
Apoptosis and Autophagy Regulation
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Reagents Dulbecco's modified Eagle's medium (DMEM), trypsin-ethylenediaminetetraacetic acid, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were obtained from Biowest (Nuaillé, France). Polyvinylidene difluoride (PVDF) membranes, goat anti-rabbit, and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Millipore (Billerica, MA, USA). Protease inhibitor cocktail, DMSO, salubrinal (Sal), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ROS Green Flow Cytometry Assay Kit, Z-DEVD-FMK (caspase-3 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), 3-methyladenine (3-MA), and rabbit anti-human β-actin antibodies were obtained from Sigma (St. Louis, MO, USA). Cell extraction RIPA buffer was obtained from TOOLS (Taiwan). Enhanced chemiluminescence (ECL) Western blotting reagents were obtained from Pierce Biotechnology (Rockford, IL, USA). Antibodies against Beclin-1, LC3-I, LC3-II, Atg3, Atg5, Atg7, Atg12, and Atg16 were obtained from Epitomics (Burlingame, CA, USA). Antibodies against procaspase 3, cleaved-caspase 3, procaspase 9, cleaved-caspase 9, cytochrome c, Bax, Bad, p-Bad, Bcl-2, Bcl-xl, Mcl-1, GRP78, CALR, elF2α, p-elF2α, IRE1α, PERK, p-PERK, ATF4, ATF6-f, CHOP, and PARP1 were obtained from Cell Signaling Technology (Danvers, MA, USA).
9
Western Blot Analysis of Liver Proteins
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Liver tissues were homogenized and sonicated in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitor cocktail and phosphostop (Roche Diagnostics, Indianapolis, IN), and total protein was extracted by centrifugation. A total of 30 μg protein was resolved on 4–12% precast SDS-PAGE gel (Invitrogen, Grand Island, NY) and transblotted onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). Membranes were probed with antisera against p-JNK, JNK, p-PDK, PDK, p-AKT, AKT, p-PKCζ, PKCζ, p-PERK, PERK, p-elF2α, elF2α, p-IRE-1, IRE-1, GRP78, CHOP, caspase-3, cleaved caspase-3, ATF-6α, XBP-1, BCL-2, Bax, LC-3B and β-actin (Cell Signaling Technology, Danvers, MA). Isotype-matched horseradish peroxidase conjugated secondary antibodies, enhanced chemiluminescence substrate (Pierce, Rockford, IL) and a FluorChem 8900 digital imaging system (AlphaInnotech, San Leandro, CA) were used to visualize protein bands. Densitometric analyses was performed with VisionWorks® Software, version 6.8 (UVP, Upland, CA).
10
Investigating F508del CFTR Ubiquitination and Degradation
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CFTR antibody 217 was purchased from the University of North Carolina at Chapel Hill. Antibodies targeting HA and Myc epitope, BIP, CHOP, PERK, CNX, PDI, P-elf2α, elf2α, ubiquitin, and K63, K48, K33, and actin antibodies were purchased from Cell Signaling. HRP-secondary antibodies were from Abcam.
F508del in the pcDNA3.1 vector was described previously 43 (link) . The N-terminal HA-tagged ubiquitin was ampli ed from human cDNA (Invitrogen) and subcloned into pRK5, which was used as a template for other Ub mutants for different lysine mutations using the site-directed mutagenesis kit (Stratagene).
pcDNA3.1-FLAG-RNF5, pcDNA3.1-FLAG-RNF5CS were from Dr. Cyr 19 , pGEX6P-GST-Rad23a, pGEX6P-GST-Adrm1, and pGEX6P-GST-S5a were from Addgene. All plasmids were con rmed by DNA sequencing. MG-132, Cycloheximide, IPTG, and tunicamycin were from Sigma. Glutathione Sepharose 4B was from Fisher Scienti c. TRIzol reagent was purchased from Invitrogen.
Cell culture and transfection HEK293 cells were cultured in Dulbecco's modi ed Eagle's (DMEM, Invitrogen) supplemented with 10 % fetal bovine serum. CFBE-F508del cell was cultured in MEM (Gibco) with 10% fetal bovine serum and 1 µg/ml puromycin. All cells were maintained in a humidi ed atmosphere containing 5 % CO 2 at 37 °C.
Cells were transiently transfected using Lipofectamine 3000 (Invitrogen) and harvested at 48 hours posttransfection.
F508del in the pcDNA3.1 vector was described previously 43 (link) . The N-terminal HA-tagged ubiquitin was ampli ed from human cDNA (Invitrogen) and subcloned into pRK5, which was used as a template for other Ub mutants for different lysine mutations using the site-directed mutagenesis kit (Stratagene).
pcDNA3.1-FLAG-RNF5, pcDNA3.1-FLAG-RNF5CS were from Dr. Cyr 19 , pGEX6P-GST-Rad23a, pGEX6P-GST-Adrm1, and pGEX6P-GST-S5a were from Addgene. All plasmids were con rmed by DNA sequencing. MG-132, Cycloheximide, IPTG, and tunicamycin were from Sigma. Glutathione Sepharose 4B was from Fisher Scienti c. TRIzol reagent was purchased from Invitrogen.
Cell culture and transfection HEK293 cells were cultured in Dulbecco's modi ed Eagle's (DMEM, Invitrogen) supplemented with 10 % fetal bovine serum. CFBE-F508del cell was cultured in MEM (Gibco) with 10% fetal bovine serum and 1 µg/ml puromycin. All cells were maintained in a humidi ed atmosphere containing 5 % CO 2 at 37 °C.
Cells were transiently transfected using Lipofectamine 3000 (Invitrogen) and harvested at 48 hours posttransfection.
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