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.
Total eif2α
Manufactured by Cell Signaling Technology
Sourced in United States
Total eIF2α is a lab equipment product that measures the total amount of eIF2α (eukaryotic translation initiation factor 2 subunit alpha) in a sample. eIF2α is a key component of the eukaryotic protein synthesis machinery. This product provides a quantitative assessment of the total eIF2α levels in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Phospho eif2α, by Cell Signaling Technology (11 mentions)
P eif2α, by Cell Signaling Technology (8 mentions)
β actin, by Merck Group (8 mentions)
Phospho eif2α ser51, by Cell Signaling Technology (5 mentions)
Phosphorylated eif2α, by Cell Signaling Technology (4 mentions)
Total p38, by Cell Signaling Technology (4 mentions)
Phospho p38, by Cell Signaling Technology (4 mentions)
Western lightening plus ecl, by PerkinElmer (4 mentions)
Tunicamycin, by Merck Group (3 mentions)
Spliced xbp1, by BioLegend (3 mentions)
P erk, by Cell Signaling Technology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Total perk, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ab32157, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Image studio lite quantification software, by LI COR (2 mentions)
Xbp1s, by Cell Signaling Technology (2 mentions)
Cyp1a1, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (2 mentions)
39 protocols using total eif2α
1
Elucidating eIF2α-PHLPP Interaction
2
Phosphorylated eIF2α analysis in Plasmodium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pf3D7 cultures were subjected to two rounds of tight synchronization within a span of 3 h in the previous cycle, followed by one additional synchronization in the subsequent cycle immediately after RBC invasion. After 3 h post-invasion, ring stage parasites were treated for 6 h with 250 μM MSO in RPMI-gln medium. For eIF2α phosphorylation analysis in PbWT and GSKO parasites, infected mouse blood of similar parasitemia predominantly containing the ring stages was incubated in vitro with 250 µM MSO for 6 h in RPMI-gln medium. The parasite pellets were then prepared by saponin treatment and lysed with 0.5x PBS containing 0.5% Triton X-100, Halt protease inhibitor and PhosSTOP (Roche, 4906845001). Western blot analysis was performed with phosphorylated eIF2α (#3398, Cell Signaling Technology) and total eIF2α (#9722, Cell Signaling Technology) antibodies.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
4
Comprehensive Antibody Panel for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following commercial primary antibodies were used: G3BP1 (mouse monoclonal; BD Biosciences); TIAR (mouse monoclonal; BD Biosciences); NONO (rabbit polyclonal C-terminal; Sigma-Aldrich); SFPQ (rabbit monoclonal; ab177149, Abcam); CPSF6 (rabbit polyclonal; A301-356A, Bethyl); FUS (mouse monoclonal; 4H11, Santa Cruz); ELAVL1 (rabbit polyclonal, 11910-1-AP, Proteintech); PABPC1 (rabbit polyclonal, Cell Signaling, 4992); EIF4E (mouse monoclonal; BD Biosciences); UBAP2L (rabbit polyclonal; A300-533A, Bethyl); DAZAP1 (rabbit polyclonal; A303-984A, Bethyl); hnRNP K (rabbit polyclonal; A300-674A, Bethyl); PSPC1 (rabbit polyclonal N-terminal; Sigma-Aldrich); TDP-43 (rabbit polyclonal C-terminal; Sigma-Aldrich); HNRNPA3 (rabbit polyclonal; 25142-1-AP, Proteintech); RBM12B (rabbit polyclonal; 17137-1-AP, Proteintech); SRSF9 (rabbit polyclonal; 17926-1-AP, Proteintech); SMARCA5 (rabbit polyclonal; 13066-1-AP, Proteintech); MATR3 (rabbit polyclonal; 12202-2-AP, Proteintech); YBX1 (rabbit polyclonal; 20339-1-AP, Proteintech); GFP (mouse monoclonal; sc-9996, Santa Cruz); Flag (DYKDDDDK tag, mouse monoclonal; 9A3, Cell Signaling); eIF2α phosphorylated at Ser51 (rabbit monoclonal; ab32157, Abcam); total eIF2α (rabbit monoclonal; D7D3, Cell Signaling); and β-actin (mouse monoclonal; A5441, Sigma-Aldrich). Antibodies were used at 1:1,000 dilution for all applications unless stated otherwise.
5
Quantitative Western Blot Analysis of Liver Proteins
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were denatured by boiling in SDS sample buffer containing 10% β-mercaptoethanol and then subjected to 10% SDS-PAGE. Blots were transferred onto a nitrocellulose membrane and blocked with Blotting Grade Blocker (#170-6404, Bio-Rad, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membrane, then immunoblotted with appropriate primary antibodies. Membranes were incubated overnight with the following primary antibodies: p-eIF2α (1:1000; #3398, Cell Signaling, Danvers, MA, USA); total eIF2α (1:1000; #2103, Cell Signaling); Tubulin (1:40 000; #SAB4500087, Sigma-Aldrich) Puromycin (1:10 000; #MABE342, Millipore, Burlington, MA, USA); and ß-actin (1:12 000; #sc1616, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% BSA in TBST at 4°C and subsequently washed thrice for 5 min with TBST. Incubation with the HRP-conjugated secondary antibodies goat anti-rabbit, goat anti-mouse or rabbit anti-goat (1:10 000; Jackson Immunoresearch, West Grove, PA, USA) was done for 1 h at room temperature. Immunodetection was carried out with EZ-ECL kit (#20500120, Biological Industries, Beit Haemek, Israel), acquired with Chemi XRS Gel Doc (Bio-Rad), and quantified using the Quantity One software (Bio-Rad). Intensity was normalized to background signal.
7
Cellular Protein Extraction and Analysis
8
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of protein lysates were separated by SDS-PAGE, and immunoblot analyses were carried out for three independent experiments using horseradish peroxidase–tagged secondary antibody. Primary antibodies used for immunoblots included total eIF2α (Cell Signaling Technology, #9722), eIF2α-P (Cell Signaling Technology, #9721), custom affinity-purified ATF4 antibody [26 (link)], CAT1 (Abcam, ab37588), GAPDH (Abcam, ab9485), puromycin (EDM Millipore, #17H1), and p70 S6 Kinase (49D7) rabbit mAb (Cell Signaling Technology #2708). Blots were incubated with Pierce ECL Western Blotting Substrate prior to imaging on FluorChem M- Multiplex fluorescence (Protein Simple).
9
Western Blot Analysis of Protein Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were harvested in 2% sodium dodecyl sulfate (SDS) and equal amounts of lysates were separated on a 10% SDS-polyacrylamide gel (except for Fig 7 , where a 15% SDS-polyacrylamide gel was used) and transferred to polyvinylide difluoride (PVDF) membranes. Proteins were detected using the Western-Star chemiluminescent detection system (Applied Biosystems) with the following primary antibodies: PKR D7F7 (#12297, Cell Signaling Technology), P-PKR E120 (ab32036, abcam), Penta-His (34660, Qiagen), K3L (a gift from J. Tartaglia [37 (link)]), P-eIF2α (#3597, Cell Signaling), total eIF2α (#2103, Cell Signaling), and Actin (A2066, Sigma).
10
Immunoblot Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot analysis was performed following our published standard procedures. The following primary antibodies were used: CYP1A1 (sc-20772, Santa Cruz Biotechnology), total p38 (#9212, Cell Signaling, Danvers, MA), phospho-p38 (#9211, Cell Signaling), total eIF2α (#9722, Cell Signaling), phospho-eIF2α (#9721, Cell Signaling), HO-1 (#5853, Cell Signaling). Use of HRP-conjugated goat anti-rabbit (111-035-144) or goat anti-mouse secondary antibody (115-035-146, Jackson Immunological Research) was followed by visualization using enhanced chemiluminescence detection reagents.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!