P eif2α eif2α

Manufactured by Cell Signaling Technology

Sourced in United States

P-eIF2α/eIF2α is a quantitative sandwich immunoassay designed for the measurement of phosphorylated eIF2α (Ser51) and total eIF2α protein levels in cell and tissue lysates. The assay utilizes capture and detection antibodies specific for phosphorylated eIF2α and total eIF2α.

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2 protocols using p eif2α eif2α

Western Blot for Protein Expression Analysis

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Western blot analysis was performed using protein extracts of HBMECs. Proteins were separated by electrophoresis and transferred to PVDF membranes. The membranes were blocked with either 5 % nonfat dry milk or 5 % BSA in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h to block nonspecific immunoreactivity. Subsequently, the membranes were incubated with the corresponding primary antibodies overnight at 4 °C. After four washes in TBST, the membranes were incubated with secondary antibodies for 1 h and detected by the enhanced chemiluminescence detection kit. In some cases, the blots were stripped and reprobed with antibodies specific for Bax, Bcl-2, cytochrome c, BiP/GRP78, p-PERK/PERK, p-IRE1/IRE1 (Santacruz Bio-technology, 1:500), p-eIF2α/eIF2α, caspases 12, and CHOP/AGDD153 (Cell Signaling, Danvers, MA). All of the westerns were repeated at least three times.

Ma R., Yang L., Niu F, & Buch S. (2014). HIV Tat-Mediated Induction of Human Brain Microvascular Endothelial Cell Apoptosis Involves Endoplasmic Reticulum Stress and Mitochondrial Dysfunction. Molecular neurobiology, 53(1), 132-142.

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Comprehensive Protein Extraction and Analysis

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Protein extraction was performed by lysing the cells in RIPA (radio-immunoprecipitation assay) buffer. The protein concentration was determined using the DC protein assay, and equal amounts of protein were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked with 5% nonfat milk in TBST for 1 h. The primary antibodies were added and incubated overnight at 4 °C with shaking. The membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. The primary antibodies of TTl1, FGFR3, α-actin, p53, p21, ATF3, FAS, TOM20, mtTFA, and PGC1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); TELO2, Cyclin D1, and γ-H2A.x were obtained from Abcam (Cambridge, UK); and p-mTOR, mTOR, p-ATM, ATM, p-p53 (ser15), MDM2, DEC1, p-c-JUN, c-JUN, p-Akt, Akt, p-p38, p38, p-PI3K, PI3K, p-ERK, ERK, p-Chk2, Chk2, p-AMPK, AMPK, p-eIF2α eIF2α, p-P70S6K, P70S6K, p-TSC2, TSC2, and 4EBP1 were obtained from Cell Signaling (Danvers, MA, USA).

Feng S.W., Wu Z.S., Chiu Y.L, & Huang S.M. (2023). Exploring the Functional Roles of Telomere Maintenance 2 in the Tumorigenesis of Glioblastoma Multiforme and Drug Responsiveness to Temozolomide. International Journal of Molecular Sciences, 24(11), 9256.

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