Cell lysates were prepared by lysis in RIPA buffer containing protease and phosphatase inhibitors and immunoblotting was performed by standard methods. The following antibodies were used: TSPO (ab109497, Abcam), ATF4(#11815, Cell signaling) and phospho eiF2a (#3398, Cell Signaling).
Phospho eif2a
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-eIF2a is a lab equipment product that detects phosphorylation of the eIF2a protein, a key regulatory subunit involved in the initiation of protein translation. This product provides a tool for researchers to study the role of eIF2a phosphorylation in cellular stress response pathways.
Automatically generated - may contain errors
Lab products found in correlation
Xbp1s, by Cell Signaling Technology (2 mentions)
Total jnk, by Cell Signaling Technology (2 mentions)
Total eif2a, by Cell Signaling Technology (2 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
Phospho perk, by Cell Signaling Technology (1 mentions)
Serca, by Cell Signaling Technology (1 mentions)
Apaf 1, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
H3k27me3, by Abcam (1 mentions)
Phospho stat3 y705, by Cell Signaling Technology (1 mentions)
Stat6, by Santa Cruz Biotechnology (1 mentions)
P stat6 y641, by Abcam (1 mentions)
Phospho histone h2ax s139, by Cell Signaling Technology (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
Histone h3, by Abcam (1 mentions)
E cadherin, by BD (1 mentions)
8 protocols using phospho eif2a
1
Cell Lysis and Immunoblotting Assay
2
Heat Stress Regulation of Apoptosis
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HUVEC cells were pretreated with or without heat stress at 43°C for 2 h, and further incubated at 37°C for different time as indicated. Western blot analysis was performed as described previously [25] (link), [26] (link) using the following antibodies: cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA (all used at 1∶1000; Cell Signaling Technology, Danvers, USA). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Zhongshan Inc, China), and signal was visualized enhanced chemiluminescence (Pierce, Rockford, IL, USA).
3
Protein Expression Analysis by Western Blot
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Tissues/cell lysate pellets were subjected to SDS-PAGE, transferred into 0.45 μm PVDF membranes and blocked 30 minutes at room temperature with 3% skim milk (Sigma-Aldrich), prior overnight incubation with the following primary antibodies: phospho-ATM S1981 and STAT6 (all from Santa Cruz Biotechnology, Dallas, TX); β-actin (Sigma-Aldrich); Vimentin, p-STAT6 Y641, Histone H3, HP1γ, and H3K27me3 (all from Abcam, Cambridge, UK); BAX, IRE1-α, phospho-EIF2A, phospho-CHK2 T387, CHOP, phospho-STAT3 Y705, and phospho-Histone H2AX S139 (all from Cell Signaling Technology); HP1β (Thermo Fischer Scientific, Waltham, MA); STAT3, BCL-2, BCL-XL, and E-cadherin (BD Biosciences, Franklin Lakes, NJ); phospho-PERK (US Biologicals, Salem, MA). After 30 min incubation with their respectively HRP-conjugated secondary antibodies (GE healthcare, Chicago, IL) membranes were developed using the SuperSignal West Pico Chemoluminescence Substrate (Thermo Fischer Scientific). Western blots were quantified using ImageJ 1.43u Software (National Institutes of Health, Bethesda, Maryland, USA. https://imagej.nih.gov/ij/ , 1997–2016).
4
Empagliflozin and Compound C Protocol
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Empagliflozin (EMPA, Cat: HY-108682) and Compound C (Dorsomorphin; Cat HY-13418A) were purchased from MedChemExpress (Monmouth Junction, NJ, United States). Other chemicals, including bovine serum albumin (BSA; 2207008), PA (P5585), and insulin (I9278) were purchased from Sigma-Aldrich (Burlington, MA, United States). 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxyglucose (2-NBDG; N13195) was obtained from Thermo Fisher Scientific (Waltham, MA, United States). Cell Death Detection enzyme-linked immunosorbent assay (ELISAplus) kit was purchased from Roche Applied Science (Roche Applied Science, Mannheim, Germany). AMP colorimetric assay kit was purchased from Biovision (Cat; K229, Milpitas, CA, United States). Antibodies targeting phospho-AKT (#9271), T-AKT (#9272), phospho-GSK3 α/β (#9331), T-GSK3β (#9315), phospho-JNK (#9251), T-JNK (#9252), phospho-eIF2A (#9721), T-eIF2A (#9722), CHOP (#2895), phospho-NF-κB p65 (#3033), NF-κB p65 (#3034), phospho-p38 (#9211), cleaved caspase-3(#9661), phospho-AMP (#2531), and T-AMP (#2532) were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-β-actin (A300-491A) antibody was purchased from Bethyl Laboratories (Montgomery, TX, United States).
5
Analyzing Cellular Signaling Pathways
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The following antibodies were purchased from Cell Signaling Technologies (Denver, CO, USA): phospho-EGFR, EGFR, p65, phospho-p65, phospho-eIF2a, eIF2a, caspase-3, and cleaved caspase-3 (Table S1 ). Antibodies to NumbL and TRAF6 were obtained from SantaCruz Biotechnology (Dallas, TX, USA). The sequence of siRNAs (mission siRNAs purchased from Sigma-Aldrich, St. Louis, MO, USA) used against NumbL was 5′-GAACUCACCUUUCAAACGU[dT][dT]-3′] and 5′-ACGUUUGAAAGGUGAGUUC[dT][dT]-3′, and that of Numb was 5′-GAAGACUGAUUUCCCAAUA[dT][dT]-3′ and 5′-UAUUGGGAAAUCAGUCUUC[dT][dT]-3′; MISSION siRNA Universal Negative Control was used as a control. Taqman probes for NumbL, Numb, and Hes-1 genes, cell culture media, RPMI-1640, and CMRL-1066 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). All the other reagents, if not indicated, were purchased from Sigma-Aldrich (St. Louis, MO, USA).
6
Immunoblotting Analysis of Stress Pathways
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Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer containing a cocktail of protease (Roche) and phosphatase inhibitors (Roche), separated by SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). The following antibodies were used: tubulin (Millipore), cleaved caspase 3 (Asp 175) (Cell Signaling), cleaved PARP (Asp 214) (Cell Signaling), total PARP (Cell Signaling), total JNK (Cell Signaling), phospho-JNK (T183/Y185) (Cell Signaling), total p38 (Cell Signaling), phospho-p38 (T180/Y182) (Cell Signaling), total MKK4 (Cell Signaling), phospho-MKK4 (Ser257/Thr261) (Cell Signaling), GRP78 (BD Biosciences), total eif2a (Cell Signaling), and phospho-eif2a (Cell Signaling), XBP-1 s (Cell Signaling), CHOP (Cell Signaling), and Kir6.2 (Santa Cruz). Quantification of western blot analysis was done using ImageJ.
7
Immunoblot Analysis of eIF2α Signaling
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For immunoblot analysis, HeLa cell cultures were placed on ice and lysed in ice-cold 1% CHAPSO, 150 mM NaCl, 20 mM K-HEPES, pH 7.5, 10% glycerol, 1 mM EDTA, 100 mM NaF, 17.5 mM B-glycerophosphate, and 1X protease inhibitor cocktail for 5 min. Lysates were TCA precipitated as noted above, washed with 100% acetone, resuspended in 0.5 M Tris, pH 11, 5% SDS, and protein concentrations determined via BCA assay (Pierce). Equivalent protein mass per sample was separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked in 10% nonfat dry milk/Tris-buffered saline and processed per antibody supplier's recommendations.
Antibodies used include eIF2a (Santa Cruz, Cat. No. sc11386; 1:500 dilution) and phospho-eIF2a (Cell Signaling Technologies, Cat. No. 3398; 1:1500 dilution).
Antibodies used include eIF2a (Santa Cruz, Cat. No. sc11386; 1:500 dilution) and phospho-eIF2a (Cell Signaling Technologies, Cat. No. 3398; 1:1500 dilution).
8
Western Blot Analysis of Cell Signaling
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Immunoblots were done as previously described (Jornayvaz et al. 2012) . Membranes were incubated overnight with primary antibodies for phospho-Akt2 (Ser 474 ) (Cell Signaling Technology, Danvers, MA, USA), phosphoenolpyruvate carboxykinase (PEPCK; Abcam, Cambride, MA, USA), pyruvate carboxylase (PC; Abcam), uncoupling protein 1 (UCP1; Santa Cruz Biotechnology), C/EBP homologous protein (CHOP; Cell Signaling Technology), IgH chain binding protein (BIP; Cell Signaling Technology), phospho-eIF2a (Cell Signaling Technology), or phospho-JNK (Cell Signaling Technology). After further washings, membranes were incubated with HRP-conjugated secondary antibody (Bio-Rad) and visualized by ECL substrate (Pierce, Rockford, IL, USA). Membranes were stripped and reblotted with anti-total Akt antibody (Cell Signaling Technology), total eIF2a (Cell Signaling Technology), total JNK (Cell Signaling Technology), or glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology). Bands were then quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
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