phosphorylated eif2α levels  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure

doi: 10.1016/j.jbc.2021.100395

Figure Lengend Snippet: Comparing signaling pathways that regulate protein synthesis in gastrocnemius muscle of WT and MKO mice . WT and MKO mice were treated with or without DEX (10 mg/kg body weight) for 1 week. At the end of treatment, mice were IP injected with insulin (1 unit per kg body weight) or PBS for 10 min, and gastrocnemius muscle was isolated and processed for ELISA to monitor the phosphorylation status of ( A ) p70S6K at Thr389, ( B ) 4E-BP1 at Thr37/Ser46 and ( C ) eIF2α at Ser52. n = 3 to 6, significance was denoted as ∗ p ≤ 0.05. Error bars represent the SD. DEX, dexamethasone.

Article Snippet: Relative total eIF2α and phosphorylated eIF2α levels were measured using the PathScan Phospho-eIF2α (Ser51) and total eIF2α Sandwich ELISA kits (Cell Signaling Technology, Catalog #7286 and Catalog #7952.)

Techniques: Protein-Protein interactions, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Aging Cell

Article Title: The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans

doi: 10.1111/acel.12101

Figure Lengend Snippet: Conservation in gene structure and function of Caenorhabditis elegans gcn-2 . (A) Gene structure and predicted protein domains of GCN-2, designed using the Prosite MyDomains ( http://prosite.expasy.org/mydomains/ ): Black boxes represent exons linked by lines corresponding to introns, and white boxes indicate the 5′ and 3′ UTR found in a second alternative isoform ( http://www.wormbase.org ). The graphic was created using the Exon-Intron Graphic Maker ( http://wormweb.org/exonintron ). Branches point to the sequences deleted in the two alleles ok871 and ok886 . Black arrowheads indicate the position of primers used in this study (Table ). (B) Reverse transcription (RT)–PCR analysis with primers G1/G2 (shown in A pane) and qRT-PCR analysis with primers G4/G5 (shown in A pane). (C) Western blot analysis showing the levels of phosphorylated (P-eIF2α) normalized by the total amount of eIF2α, in whole worm extracts. Basal levels of P-eIF2α under well-fed conditions in untreated or gcn-2(RNAi) -treated N2 and gcn-2 mutants (left pane). Induced levels of P-eIF2α under amino acid limitation in krs-1(RNAi) treated worms (right pane).

Article Snippet: After 2–3 washes to remove bacteria, they were frozen in ethanol dry ice and, before loading onto SDS-PAGE, worm pellets were boiled in 50 μL 2X SDS-sample buffer for 10 min. Primary antibodies were a polyclonal antibody raised against worm eIF2α, a kind gift from Dr. Shin Takagi (Nukazuka et al ., ), for total (T)-eIF2α and an anti-phospho-eIF2α antibody (Santa Cruz Biotechnology, sc-101670) for phosphorylated (P)-eIF2α levels.

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot