anti phospho eif2α ser51  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: A stress-induced PI3P complex controls autophagy in erythroid precursors

doi: 10.1016/j.isci.2026.115182

Figure Lengend Snippet: SAR405 inhibits autophagy during erythropoiesis (A) RT-qPCR in WT murine splenic erythroblast cells (Lineage − CD71 + ) 3 days after acute hemolytic anemia. N = 3 mice per group. Error bars represent standard error of mean (SEM). ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test). (B) RT-qPCR in murine splenic erythroblasts (lineage − CD71 + Kit + ) cells isolated from control (WT) or Berkeley Sickle Cell Disease (SCD) mice ( N = 3 per genotype). Error bars represent standard error of mean (SEM). ∗ p < 0.05; ∗∗ p < 0.01 (two-tailed unpaired Student’s t test). Target mRNA expression levels were normalized to 18S rRNA. (C) Western blotting of Lineage − CD71 + cells isolated from control (PBS-treated) and anemia (PHZ-treated) spleens for autophagy-related proteins. N = 3 WT mice per experimental group. (D) Semi-quantitative densitometry of p -Ampk (Thr172) relative to total Ampk and p -eIF2α (Ser51) relative to total eIF2α ( N = 3 mice). Error bars represent standard error of mean (SEM). ∗ p < 0.05; ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test). (E) Experimental layout of splenic EPCs isolated from Samd14 fl/fl (WT) and Samd14 fl/fl;Vav−Cre (S14-CKO) mice post PHZ-induced anemia. EPCs were retrovirally infected to express the GFP-LC3-RFP probe (Addgene #84573). After 24 h, cells were treated with VPS34 inhibitor SAR405 for an additional 48 h before analysis by flow cytometry. (F) Schematic representation of autophagic flux measurement using GFP-LC3-RFP probe. (G) Gating strategy for GFP and RFP signal measurement by flow cytometry. Progressively maturing erythroid cells (R1-R4) were gated within a viable RFP-expressing cell population using cell surface markers CD71 and TER119. (H) Quantitation of GFP/RFP median fluorescence intensity ratio calculated within R1 (CD71 low/med TER119 - ), R2 (CD71 high TER119 - ), R3 (CD71 high TER119 + ), and R4 (CD71 low/neg TER119 + ) populations in DMSO or SAR405-treated WT cultures ( n = 3 mice). Error bars represent standard error of mean (SEM). ∗∗∗ p < 0.001 (One-way ANOVA). (I) Quantitation of GFP/RFP median fluorescence intensity ratio calculated within R2 (CD71 high TER119 - ) and R4 (CD71 low/neg TER119 + ) populations in DMSO or SAR405-treated WT and S14-CKO cultures ( n = 3 mice per group). Error bars represent standard deviation (SD). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (two-way ANOVA).

Article Snippet: Anti-phospho-eIF2α (Ser51) (clone 119A11, rabbit monoclonal) , Cell Signaling Technology , Cat.: #3597; RRID: AB_390740.

Techniques: Quantitative RT-PCR, Two Tailed Test, Isolation, Control, Expressing, Western Blot, Infection, Flow Cytometry, Quantitation Assay, Fluorescence, Standard Deviation

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Inhibition of tRNA charging activates the ISR without inducing RNP granules. ( A ) U-2 OS cells were treated with 0.2% DMSO carrier, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) for 1 h, and Western blotting for phosphorylated and total eIF2α was done. Total protein ( n = 3 independent replicates) and molecular weights (in kilodaltons) are shown. ( B ) Translation activity was measured using bioorthogonal noncanonical amino acid tagging in cells treated as in A . Methionine or azidohomoalanine (AHA) was added 10 min prior to collection. The ratio of nascent to total protein is shown from n = 3 independent replicates. ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A and fixed, and nuclei were stained with Hoechst (blue). The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥305 cells were counted in each condition. Scale bars, 10 µm. ( D ) Cells were treated as in C , and immunofluorescence and fluorescence in situ hybridization were done to detect UBAP2L (magenta), PABPC1 (cyan), and poly(A) RNA with oligo(dT) probes (yellow) ( n = 2 independent experiments). ( E ) Cells were treated with 0.2% DMSO, 20 µM HF plus 0.2% DMSO, 250 µM arsenite, or 1.25 µM thapsigargin for 1 h, and the percentage of cells with SGs was quantified ( n = 3 independent experiments); ≥287 cells were counted per treatment. ( F ) Cells were treated, and immunofluorescence microscopy was done as in D to detect the P-body marker DCP1A. Quantification of the percentage of cells with P-bodies (PBs) from n = 3 independent experiments is shown; ≥364 cells were counted per treatment. ( G ) Cells were treated as in C to detect XRN1 (magenta) and EDC4 (yellow) by immunofluorescence microscopy ( n = 2 independent experiments). ( H ) U-2 OS cells were treated as in E , and immunofluorescence microscopy was done to detect DCP1A. The percentage of cells with PBs from n = 3 independent experiments is shown; ≥351 cells were counted per treatment across all replicates. Representative images are shown with the average ± SEM for each experiment, and green, gray, and pink points represent the average of each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: eIF2α , Santa Cruz Biotechnology , SC-133132 , 1:1000 (Western).

Techniques: Inhibition, Western Blot, Activity Assay, Stable Transfection, Expressing, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Microscopy, Marker

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Ribosome collisions contribute to ISR activation during halofuginone treatment. ( A ) Cells were treated with 0.2% DMSO, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) with or without 10 µg/mL puromycin (Puro) for 1 h, and P-eIF2α and total eIF2α levels were determined by Western blotting. Quantification of P-eIF2α:eIF2α is shown at the right , and a representative blot with total protein from n = 3 independent experiments shown at the left . ( B ) Western blotting of total and P-eIF2α was done from wild-type (WT) and ZNF598 knockout (KO) cells treated with 20 µM HF for 0.5, 1, or 4 h. A representative blot with total protein is shown ( top ), and results from n = 3 independent replicates were quantified ( bottom ). ( C ) Western blotting of P-eIF2α and total eIF2α from WT and ZNF598 KO cells treated with 20 µM HF for 1 or 4 h in the absence or presence of 10 µg/mL puromycin added 1 h prior to collection. Representative blot with total protein from n = 3 independent experiments ( top ) and quantification of P-eIF2α:eIF2α ( bottom ) are shown. ( D ) Wild-type and ZNF598 KO cells were treated as in B , and Western blotting for GADD34, CReP (marked by an asterisk), and β-tubulin was done with quantification relative to total protein. Representative blots are shown at the top , and results from n = 3 independent experiments are shown below . Molecular weights (in kilodaltons) are indicated on each blot. Quantification is reported as average ± SEM, with pink, gray, and green points representing each replicate. Statistical significance was determined with ordinary one-way ANOVAs and Tukey's multiple comparisons tests. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: eIF2α , Santa Cruz Biotechnology , SC-133132 , 1:1000 (Western).

Techniques: Activation Assay, Western Blot, Knock-Out

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: tRNA synthetase inhibition causes persistent ribosome stalls. ( A ) U-2 OS cells expressing GFP-G3BP1 were treated with 20 µM halofuginone (HF) or 0.2% DMSO carrier. Images were collected every 2 h for 16 h and representative images are shown ( n = 3 independent experiments). ( B ) Cells were treated with DMSO (−) or 20 µM HF for 4 or 16 h, and P-eIF2α and total eIF2α were detected by Western blotting. Representative blots with total protein are shown at the left , and the average ± SEM of n = 3 independent experiments is shown at the right . Molecular weights (in kilodaltons) are shown. ( C ) Cells were treated with 20 µM HF for 1 h (black line), 4 h (purple line), or 16 h (green line), and polysome profiling was performed. A representative profile is shown at the left , and the average ± SEM of the polysome:monosome ratio from n = 3 independent replicates is shown at the right . Pink, gray, and green dots represent results from each replicate. ( D ) Cells were treated with either 0.2% DMSO carrier or 20 µM HF in the presence or absence of 250 µM sodium arsenite (As) or 2 µM rocaglamide A (RocA), and GFP-G3BP1 was imaged every 0.5 h for 3 h. Representative images are shown at the left with the average ± SEM. The percentage of cells with SGs from n = 3 independent experiments is shown at the right ; ≥288 cells were counted per treatment across all replicates. ( E ) Cells were treated with 0.2% DMSO (black line), 2 µM RocA (green line), or 2 µM RocA plus 20 µM HF (purple line) for 3 h followed by polysome profiling. Representative profiles are shown at the left , and the average ± SEM from n = 3 independent experiments is shown at the right , with pink, gray, and green dots representing each replicate. ( F ) Cells were untreated (DMSO carrier) or treated for 24, 4, or 1 h with 20 µM anisomycin (ANI) or 20 µM halofuginone, and Western blotting for p-p38 and p-JNK was done, with the total protein shown below . A representative blot of n = 2 independent experiments is shown. Scale bars, 10 µm. Significance was assessed with ordinary one-way ANOVAs and Tukey's multiple comparisons tests. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

Article Snippet: eIF2α , Santa Cruz Biotechnology , SC-133132 , 1:1000 (Western).

Techniques: Inhibition, Expressing, Western Blot

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Amino acid deprivation activates the integrated stress response without inducing stress granules. ( A ) U-2 OS cells were placed in media without arginine (−Arg) for 1, 4, or 16 h or in the same media with arginine added back (+Arg) for 4 h, and a representative Western blot for total and P-eIF2α with total protein is shown. ( B ) As in A , with glutamine (Gln)-free media or with glutamine added back (+Gln). ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A , followed by immunofluorescence for P-body (PB) marker DCP1A (magenta), and nuclei were stained with Hoechst (blue). The percentages of cells with stress granules (SGs) and PBs were quantified from n = 3 independent experiments; ≥410 cells were counted for each condition. ( D ) As in C but with glutamine deprivation; ≥312 cells were counted for each condition. ( E ) Cells were treated with DMSO or 40 nM thapsigargin (Tg) for 1 h or starved of arginine or glutamine for 4 h followed by Western blotting for total or P-eIF2α, with total protein shown below . ( F ) GFP-G3BP1-expressing U-2 OS cells were treated with DMSO control or 40 nM Tg for 1 h, and immunofluorescence was done for DCP1A. The average percentage of cells with SGs or PBs is shown at the right ; ≥440 cells were counted per treatment. Scale bars, 10 µm. Molecular weights (in kilodaltons) are shown on each blot. Images are shown for each experiment ( n = 3 independent replicates), with quantifications reported as average ± SEM and with green, gray, and pink points representing the value for each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test for all but F , where a t -test was used. (*) P < 0.05, (**) P < 0.01, (****) P < 0.001.

Article Snippet: eIF2α , Santa Cruz Biotechnology , SC-133132 , 1:1000 (Western).

Techniques: Western Blot, Stable Transfection, Expressing, Immunofluorescence, Marker, Staining, Control

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Model depicting the requirement of tRNA synthetase activity for RNP granule assembly. Upon tRNA synthetase inhibition, ribosomes stall, and eIF2α phosphorylation results from uncharged tRNAs and collided ribosomes. Ribosome-associated quality control (RQC) clears stalled, collided ribosomes but cannot resolve ribosomes that stall in the absence of collisions. The association of stalled ribosomes with mRNAs blocks the assembly of stress granules and P-bodies.

Article Snippet: eIF2α , Santa Cruz Biotechnology , SC-133132 , 1:1000 (Western).

Techniques: Activity Assay, Inhibition, Phospho-proteomics, Control