Cellular protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.1% SDS). Protein lysates were subjected to immunoblotting using antibodies against SOD1, poly ADP ribose polymerase (PARP, BD Bioscience Pharmingen, San Diego, CA), caspase-3, caspase-8, p53, p21, MCL-1, BclxL, c-Myc, cytochrome-c (Santa Cruz Biotechnology), caspase-9, p-eIF2α (Abcam, Cambridge, MA), cyclin-B1, CDC25C, CDC2, HSP60, CLPP, COX IV, PERK, BIP, Calnexin, GFP (Cell Signaling, Beverly, MA), polyubiquitin (Enzo Life Sciences, Inc., Farmingdale, NY), GAPDH, or β-actin (Sigma-Aldrich).
P eif2α
Manufactured by Abcam
Sourced in United States, United Kingdom
P-eIF2α is a protein that is phosphorylated on the eIF2α subunit of the eukaryotic translation initiation factor 2 (eIF2) complex. The phosphorylation of eIF2α plays a key role in the regulation of protein synthesis in response to various cellular stresses.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (7 mentions)
Eif2α, by Cell Signaling Technology (7 mentions)
Gapdh, by Cell Signaling Technology (5 mentions)
Eif2α, by Abcam (5 mentions)
Bcl2 associated x (bax), by Abcam (4 mentions)
Grp78, by Abcam (4 mentions)
β actin, by Abcam (4 mentions)
Ire1α, by Abcam (4 mentions)
P erk, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Beclin 1, by Abcam (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
P pkr, by Abcam (3 mentions)
Caspase 9, by Abcam (2 mentions)
Cyclin b1, by Cell Signaling Technology (2 mentions)
Cdc25c, by Cell Signaling Technology (2 mentions)
Calnexin, by Cell Signaling Technology (2 mentions)
Caspase 8, by Santa Cruz Biotechnology (2 mentions)
Caspase 3, by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Abcam (2 mentions)
32 protocols using p eif2α
1
Immunoblotting Analysis of Cellular Proteins
2
Podocyte Immunofluorescence and F-actin Staining
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Podocytes were cultured on coverslips in 24-well plates for 24 h. Cells were washed 3-times with PBS, fixed with 4% paraformaldehyde on ice for 10 min, permeabilized with 0.1% Triton X-100 (Dingguo) for 15 min, and blocked with 10% normal goat serum for 1 h at room temperature. Podocytes were then incubated with the primary antibodies at 4°C overnight, washed with PBS, and incubated with Cy3- or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibodies (Beyotime, Shanghai, China) in the dark for 1 h at 37°C. The nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI, Genview) for 5 min in the dark. Finally, the coverslips were mounted on slides with an anti-fluorescence quencher, and the stained podocytes were observed with a Leica DM60008 microscope. The following primary antibodies were used: GRP78 antibody (1:500, Abcam) and phosphorylated eukaryotic initiation factor 2α (P-eIF2α) (1:500, Abcam).
Alternatively, podocytes were permeabilized with Triton X-100 and washed 3-times with PBS. Subsequently, podocytes were stained with 100 μg/mL phalloidin-conjugate working solution (AAT Bioquest, Sunnyvale, CA, USA) for 60 min at room temperature. Cells were washed 3-times with PBS and observed with a laser scanning confocal microscope (TCS SP8 STED, Leica, Wetzlar, Germany).
Alternatively, podocytes were permeabilized with Triton X-100 and washed 3-times with PBS. Subsequently, podocytes were stained with 100 μg/mL phalloidin-conjugate working solution (AAT Bioquest, Sunnyvale, CA, USA) for 60 min at room temperature. Cells were washed 3-times with PBS and observed with a laser scanning confocal microscope (TCS SP8 STED, Leica, Wetzlar, Germany).
3
Protein Expression Analysis in dOPCs
4
Western Blot Analysis of MSCs
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Proteins (30 μg) in the cell lysate of healthy-MSCs and CKD-MSCs were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to an 8 μm pore size nitrocellulose membrane. The membrane was blocked using 5% skim milk prepared in TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% (v/v) Tween 20) for 1 h at room temperature. The membrane was then treated with primary antibodies. The antibodies used were directed against p-PERK, anti-PERK, p-JNK, JNK, MFN1, PrPC, and β-actin (all 1:300 dilution, all from Santa Cruz Biotechnology, Dallas, TX, USA), p-eIF2α, eIF2α, ATF4, p-DRP1 (both 1:1000 dilution, both from Abcam, Cambridge, UK), p-IRE1α, IRE1α, CHOP, OPA1, and PGC-1α (all 1:1000 dilution, all from Novus, Centennial, CO, USA). Next, each membrane was washed twice, and the primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology). The protein bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
5
Western Blot Analysis of Protein Markers
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Total protein from pancreatic tissue was obtained by RIPA lysis buffer with 1% phenylmethylsulfonylfluoride (Solarbio). The proteins were electrophoresed on 10% and 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After being blocked in 5% skimmed milk for 2 h at 25 °C, the membranes were cut prior to hybridisation with primary antibodies. After that, the membranes were incubated at 4 °C overnight with primary antibodies: GPX4 (1:1000; BOSTER), xCT (1:1000; Abcam), ACSL4 (1:1000; Abcam), LPCAT3 (1:500; ABclonal, Wuhan, China), binding immunoglobulin protein (Bip, 1:1000; Zenbio, Chengdu, China), C-EBP homologous protein (CHOP, 1:1000; BOSTER, Wuhan, China), eukaryotic initiation factor 2α (EIF2α, 1:2000; Proteintech, Wuhan, China), phosphoration of EIF2α (p-EIF2α, 1:1000; Abcam), and β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA). Subsequently, the membranes were incubated with a secondary goat anti-rabbit antibody (1:10,000; Cell Signaling Technology) for 2 h at 25 °C. Protein bands were visualized using the Odyssey Fc Imaging System, and band images were analyzed using Image J software. All initial western blotting bands in this study are presented in the Supplementary Information .
6
Sorafenib-Induced Apoptosis Mechanisms
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The compound was obtained as described previously. They were dissolved in dimethyl sulfoxide (DMSO, Biosharp, Hefei, China) and stored at −20 °C. Sorafenib, 4,6-diamidino-2-phenylindole (DAPI), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, anti-rabbit IgG, and anti-mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified essential medium (DMEM), phosphate-buffered saline (PBS), trypsin-EDTA, and fetal bovine serum (FBS) were bought from GIBCO BRL. The Annexin V-FITC/PI apoptosis detection kit was purchased from Nanjin KeyGen Biotech (Nanjing, China). The following primary antibodies were used: GRP-78, hTERT, p65, IKB, Bcl-2, Bax, cyt-c, CHOP, PERK/p-PERK, p-eIF2α, ATF4, IRE1, p-IRE1α, and β-actin were purchased from Abcam Technology (Abcam, Cambridge, UK).
7
Western Blot Analysis of Hepatic Proteins
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Mouse livers and HepG2 cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA), and the protein extract was measured using the Bradford assay (Bio-Rad, 162-0115, Hercules, CA). Equal amounts of proteins (50 μg) were heat-denatured in 4× sample buffer (2% sodium dodecyl sulfate, 62.5 mM Tris (pH 6.8), 0.01% bromophenol blue, 1.43 mM mercaptoethanol, and 0.1% glycerol), separated on 10% or 12% sodium dodecyl sulfate-polyacrylamide gels, and electroblotted onto a nitrocellulose membrane (Bio-Rad). Membranes were subsequently treated with the appropriate antibodies against the following proteins: betatrophin (cat#TA326696, OriGene Technologies, Rockville, MD), phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α; cat#ab32157, Abcam, Cambridge, MA), Activating transcription factor 6 (ATF6; cat# MAB0082, Abnova, Taipei city, Taiwan), NF-κB (p65, cat#sc-372, Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (cat#a5441, Sigma-Aldrich).
8
Antibodies and Reagents for ER Stress Analysis
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9
Western Blot Analysis of eIF2α Phosphorylation
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Equal volumes (5 μl) of the in vitro reactions were loaded on 3–8% Tris-Acetate gels (ThermoFisher Scientific). Proteins were transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% skimmed milk in TBS-T (0.05% Tween) and then probed with following primary antibodies diluted in 4% BSA in TBS-T (0.05% Tween): p-eIF2α (Abcam, #ab32157, 1:1000), eIF2α (Cell Signaling, #L57A5, 1:1000), GCN2 (Cell Signaling, #E9H6C, 1:1000). Next, membranes were washed 3x in TBS-T (0.05% Tween) and incubated with appropriate secondary antibodies: Goat anti-Mouse Alexa Fluor 680 (Invitrogen, #A32729, 1:5000) or Goat anti-Rabbit Alexa Fluor 790 (Invitrogen, #A27041, 1:10,000). Proteins were visualized using the LICOR Odyssey infrared imaging system. The detection of p-eIF2α and eIF2α proteins was performed simultaneously. Bands were quantified using ImageStudioLite and analyses were performed using GraphPad software.
10
Western Blot Analysis of Cellular Stress
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Cell lysates were obtained in cell lysis buffer [50 mmol/L Tris, HCl (pH 7.4), 150 mmol/L NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate and protease/phosphatase inhibitors (Complete, Roche Diagnostics, Risch-Rotkreuz, Switzerland)], and total protein concentration in each sample was measured using a Bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies were as follows: p-eIF2α (Abcam, Cambridge, England, ab32157), eIF2α (Cell Signaling technology, Danvers, MA, USA, #9722), CHOP (Santacruz, Dallas, TX, USA, sc-575), ATF4 (Cell Signaling technology, Danvers, MA, USA, #11815), HSP70 (Enzo Life Sciences, Farmingdale, NY, USA, SPA822), cleaved Caspase-3 (Cell Signaling technology, Danvers, MA, USA, #9661), PARP (Cell signaling technology, Danvers, MA, USA, #9542), and α-tubulin (Sigma, St. Louis, MO, USA, T9026). Secondary antibodies were as follows: Peroxidase-conjugated affinipure goat anti-mouse IgG (Jackson laboratory, Bar harbor, ME, USA, 115-035-003) and anti-rabbit IgG (Jackson laboratory, Bar harbor, ME, USA, 111-035-003). Chemiluminescence detection using enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) was performed. Membranes were exposed to imaging film and developed using an automatic film processor (Agfa, Mortsel, Belgium, CP1000).
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