Fig 1: The BoHV1-activated PERK pathway facilitates viral proliferation. (A) MDBK cells were transfected with siRNAs targeting PERK for 48 h, and then cells were infected with BoHV-1. Immunoblot analysis of the silencing efficiency of PERK at 24 hpi. (B) The CCK-8 assays were performed to analyze the MDBK cell viability. (C,D) At 48 h post siRNA transfection, MDBK cells were infected with BoHV-1 (0.1 MOI). The viral titers of BoHV-1 and the relative expression of BoHV-1 gC were detected at the indicated time points. (E) The efficiency of GSK2606414 in inhibiting the PERK pathway was evaluated by detecting the ATF4 mRNA levels in MDBK cells. (F) The cytotoxicity of GSK2606414 was measured using a CCK-8 assay. (G) Immunoblot analysis of total PERK, total eIF2a, phosphorylated PERK, and phosphorylated eIF2a in MDBK cells treated with different concentrations of GSK2606414. (H,I) The viral titers and gene expression of BoHV-1 in MDBK cells treated with GSK2606414 (20 µM) or DMSO at 12 and 24 hpi were determined. (J) Effect of PERK inhibition on BoHV-1 replication stages. MDBK cells infected with BoHV-1 (0.1 MOI) were treated with GSK2606414 (20 µM) at the virus binding, virus entry, or post-entry process, respectively. The viral titers were determined in MDBK cells. The mean ± SD of data from three independent experiments are shown; * p < 0.05, ** p < 0.01.
Fig 2: Model of PERK/PKR-eIF2a negative regulation of PHEV replication. PHEV infection induces ER stress both in vitro and in vivo, and host cells sense PHEV infection and induce the activation of ATF6, IRE1, and PERK of the UPR and PKR of the ISR. Activated PERK and PKR activate eIF2a by phosphorylating serine at residue 51, and p-eIF2a negatively regulates PHEV replication by attenuating global protein translation and SG formation. The red arrows and the blunt-ended lines indicate activation and inhibition, respectively.
Fig 3: SA prevents ER stress caused by 6-OHDA-induced neurotoxicity in SH-SY5Y neuroblastoma cells. (A) Western blot analyses were performed to measure the expression levels of ER-stress-related proteins in SA-pretreated/6-OHDA-treated cells. The expression levels of total and phospho-PERK (B), total and phospho-eIF2a (C), ATF4 (D), CHOP (E), GADD45a (F), GRP94 (G), and GRP78 (H) proteins were quantified using HD imaging software. ß-actin was used as the loading control. Western blot analysis was performed in triplicate with three independent samples. Data are shown as the mean ± standard error of the mean (SEM). Significance was determined via a one-way ANOVA with a Bonferroni post hoc test. ## p < 0.01 and ### p < 0.001 vs. control group. ** p < 0.01 and *** p < 0.001 vs. the 6-OHDA-only-treated group. Cont, control; SA, sinapic acid; 6-OHDA, 6-hydroxydopamine; PERK, pancreatic ER kinase; eIF2a, eukaryotic translation initiation factor 2a; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; GADD45a, growth arrest and DNA damage-inducible protein45a; GRP94 and 78, glucose regulated protein94 and 78.
Fig 4: Oleandrin induces breast cancer ER stress.A, B MCF7 and MDA-MB-231 cells were treated with oleandrin for 0, 6, 12, and 24 h. The protein expressions were analyzed by western blotting. C MCF7 and MDA-MB-231 cells were pretreated with the PERK inhibitor GSK2606414 at 6 µM and the IRE1 inhibitor 4µ8C at 5 µM for 12 h before treatment with oleandrin for further 6 h. ß-actin was used as loading control. D Treated cells were stained with CRT and PI before detected by flow cytometry. The CRT-positive and PI-negative cells were showed in representative dot plots and quantification data. **p < 0.01. ole, oleandrin; GSK, GSK2606414.
Fig 5: The effect of PA treatment on the Notch1/Hes1 signalling pathway and ER stress in MI/R mice. Western blotting was used to detect the levels of Notch1, NICD, Hes1, ATF4, PERK, p-PERK, caspase-3 and p-caspase-3 in MI/R mouse myocardial tissue. (A) Western blot bands of the detected proteins. (B) Relative expression levels of the proteins. ***p < 0.001 vs. sham group. ??p < 0.01, ???p < 0.001 vs. MI/R group. Data are expressed as the mean ± SD.
Supplier Page from Abcam for Anti-PERK (phospho T982) antibody