eIF2 alpha Antibody - #AF6087

Figure 7. Effect of A22 on ameliorating apoptosis, ER stress, inflammation, metabolic syndrome, and fibrogenesis in HF diet-fed mice. (A) Effect of A22 on BCL-2 gene transcription. (B) Effect of A22 on BAX gene transcription. (C) Effect of A22 on expressions of apoptosis-related proteins in liver. The extracted proteins from the liver were immunoblotted with specific antibodies, and quantified based on the loading control of ACTIN. (D) Effect of A22 on ER stress. The UPR proteins (IRE-1, PERK, elF-2 and CHOP) were analyzed by using western Blot. (E) Effect of A22 on expressions of inflammatory factors. (F) Effect of A22 on expressions of fibrogenic proteins.

Fig. 5 SPI1 activates the UPRmt via PERK and inhibits chondrocyte senescence rather than by activating GCN2. a Protein expression of SPI1, PERK, p16INK4A, p21, GLB1, GCN2, eIF2α, p-eIF2α, ATF5, HSP60, LONP1 and ClpP was detected by WB in chondrocytes with SPI1 overexpression in model groups. The ratio of p-eIF2α to eIF2α is shown. b Gene expression changes of SPI1, PERK, CDKN2A, CDKN1A, GLB1, GCN2, eIF2α, ATF5, HSP60, LONP1 and ClpP was detected by RT-qPCR in chondrocytes with SPI1 overexpression in model groups. c Protein expression of SPI1, PERK, p16INK4A, p21, GLB1, GCN2, eIF2α, p-eIF2α and ATF5 were detected by WB in chondrocytes with SPI1 inhibition in model groups. The ratio of p-eIF2α to eIF2α is shown. d Gene expression changes of SPI1, PERK, CDKN2A, CDKN1A, GLB1, GCN2, eIF2α and ATF5 were detected by RT-qPCR in chondrocytes with SPI1 inhibition in model groups. The cell sample size is n = 4. Experiments were independently repeated three times. The data were expressed as mean ± SD. *P 

Figure 4. In vitro study of lactic acid-induced ER stressmediated ICD. A) Schematic diagram of ER stress-induced ICD caused by lactate accumulation. B) Western blot analysis showing the expression levels of SERCA2, PERK, p-PERK, eIF2α, and p-eIF2α ARTICLE IN PRESS in H22 cells following treatment with PBS, As³⁺, As⁵⁺, GD NPs, and GD-A NPs. C) The Ca2+-ATPase activity was assessed following the different treatment conditions (n=3). D) Western blot analysis showing the expression levels of CHOP, ATF4, GRP78 and SERCA2 in H22 cells following treatment with PBS, As³⁺, As⁵⁺, GD NPs, and GD-A NPs. E-F) Average fluorescence intensity of HMGB1 and CRT quantification in H22 cells after treatment with different groups (n=3). G) Results of extracellular ATP concentration measurements in H22 cells treated with PBS, As³⁺, As⁵⁺, GD NPs, and GD-A NPs (n=3). H) CLSM images of CRT expression in H22 cells treated with PBS, As³⁺, As⁵⁺, GD NPs, and GD-A NPs, scale bar: 20 μm. I) CLSM images of HMGB1 expression in H22 cells after treatment with PBS, As³⁺, As⁵⁺, GD NPs, and GD-A NPs, scale bar: 20 μm. J) Schematic diagram of H22 cell culture medium co-incubated with GD-A NPs and BMDCs for 24 h to induce maturation. K-M) Flow cytometric evaluation of CD80⁺/CD86⁺ expression in CD11c⁺ BMDCs following treatment with PBS, As³⁺, As⁵⁺, GD NPs, or GD-A NPs. (n=3). Results are expressed as mean ± SD; differences from the control group are denoted by * p

Fig. 5 BA triggers breast cancer cells apoptosis via ER stress-mediated pathway. a MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of BA for 24 h, and the protein levels of ER stress-associated signals were stimulated by BA in a dose-dependent manner, including GRP78, p-PERK/PERK, p-eIF2α/eIF2α, CHOP, and caspase-12. b MCF-7 and MDA-MB-231 cells were treated with BA alone or in combination with taxol for 24 h, the expression levels of GRP78, p-PERK/PERK, p-eIF2α/eIF2α, CHOP, and caspase-12 were also significantly upregulated following drug administration, especially in the co-treatment group, indicating the ER stress-mediated apoptosis pathway was aggravatedly activated by drug combination.

Fig. 1. Serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) knockdown (3cKD) in adipocytes enhanced endoplasmic reticulum overoxidation and endoplasmic reticulum stress (ERS), and promoted the expression of pro-inflammatory cytokines and adipocyte apoptosis under lipotoxicity injury. (A) Western blotting of Serpina3c protein level in 3cKD 3T3-L1 adipocytes and its control LV3 group, as well as Serpina3c overexpression (3cOV) 3T3-L1 adipocytes and its control LV5 group. (B) Cell counting kit-8 (CCK8) assay measured the cell viability (%) of LV3 group and 3cKD group after 500 μM palmitic acid (PA)treated for 24 or 48 hours. (C-J) In these experiments, LV3, 3cKD, LV5, and 3cOV groups were treated by 500 μM PA for 48 hours. (C, D) The relative mRNA levels of indicated genes in 3T3-L1 adipocytes. (E) The protein levels of indicated genes in 3T3L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (F) Hydrogen peroxide (H2O2) level in 3T3-L1 adipocytes was determined. (G) The protein levels of ERS makers in 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (H) Reactive oxygen species (ROS) level in 3T3-L1 adipocytes was detected. (I) The protein levels of mitogen-activated protein kinase signaling pathway in 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (J) Caspase-3 activity in 3T3-L1 adipocytes was determined. Data were presented as mean±standard error of the mean (n=5 for each group unless otherwise mentioned). NS, no significance; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-X-C motif chemokine ligand 1 or 10; HIF1α, hypoxia-inducible factor 1α; Ero1α, endoplasmic reticulum oxidoreductase 1α; PDIA3 or PDIA4, protein disulfide isomerase family A member 3 or 4; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein; p-eIF2α, phosphorylated eukaryotic initiation factor 2α; c-ATF6, cleaved activating transcription factor 6; XBP1S, spliced X-box binding protein 1; p-JNK, phosphorylated c-Jun N-terminal kinase; p-ERK, phosphorylated extracellular signal-regulated kinase.