Journal: Experimental & molecular medicine

Article Title: Sestrin2 protects against cholestatic liver injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-mediated pyroptosis.

doi: 10.1038/s12276-022-00737-9

Figure Lengend Snippet: Fig. 3 Bile acids upregulate Sestrin2 expression via an ATF4- and C/EBP-β-dependent mechanism. A, B HepG2 cells were treated with 200 µM CDCA or 750 µM CA for the indicated times. Cell lysates were immunoblotted with anti-p-eIF2α, anti-eIF2α, and anti-ATF4 antibodies. C Immunofluorescence staining of PDI (green) in HepG2 cells treated with 200 µM CDCA or 750 µM CA for 9 h (n = 3–4). Nuclei were stained with DAPI (blue). Scale bars, 10 μm. D, E Liver tissues were collected from mice 3 days after sham or BDL surgery (n = 4–5 mice per group) and analyzed by immunoblotting with the indicated antibodies. Band intensities were quantified and normalized to GAPDH or total protein intensities. F Immunohistochemical analysis of BiP in liver tissues from mice 3 days after sham or BDL surgery (n = 4–5 mice per group). The boxed areas are magnified in the bottom panels. Scale bars, 50 μm; 10 μm (insets). G, H HepG2 cells were infected with lentiviruses expressing shRNAs targeting luciferase (sh-Luc) or ATF4 (sh-ATF4) and treated with 200 µM CDCA or 750 µM CA for 9 h. Cell lysates were immunoblotted with anti-Sestrin2 and anti-ATF4 antibodies. I, J HepG2 cells were infected with lentiviral sh-Luc or sh-C/EBP-β and treated with 200 µM CDCA or 750 µM CA for 9 h. Cell lysates were immunoblotted with anti-Sestrin2 and anti-C/EBP-β antibodies. GAPDH or β-actin served as loading controls. Numbers below the immunoblot bands indicate fold changes normalized to the control band intensities. The data are representative of one (D–F) or at least three (A–C, G–J) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test).

Article Snippet: Immunoblotting was performed using antibodies against human Sestrin2 (Proteintech Group, USA), mouse Sestrin2 (Dr. Jun Hee Lee, University of Michigan); cleaved caspase-3, phospho-eIF2α, PERK, phospho-p70 S6 kinase, p70 S6 kinase, phospho-S6 ribosomal protein, S6 ribosomal protein, phospho-AMPK, AMPK, TSC2, NLRP3 (Cell Signaling Technology, USA), ATF4, C/EBP-β, eIF2α, caspase-1, ASC (Santa Cruz Biotechnology, USA); GSDMD (Abcam), GAPDH (Aviva Systems Biology, USA), and β-actin (Developmental Studies Hybridoma Bank, University of Iowa).

Techniques: Expressing, Staining, Western Blot, Immunohistochemical staining, Infection, Luciferase, Control

Journal: The Journal of Biological Chemistry

Article Title: Autoantibodies against the cell surface–associated chaperone GRP78 stimulate tumor growth via tissue factor

doi: 10.1074/jbc.M117.799908

Figure Lengend Snippet: Treatment with anti-GRP78 AutoAbs increases the expression of TF and UPR markers in DU145 human prostate cancer cells. A, flow cytometry analysis of csGRP78 amounts in DU145 cells. B, pathological doses of anti-GRP78 AutoAbs (60 μg/ml) increase protein expression of both TF and markers of UPR activation (PDI, IRE1, and phospho-eIF2α), compared with non-treated (0 μg/ml) cells or cells treated with a normal dose of anti-GRP78 AutoAbs (5 μg/ml). β-Actin was used as a loading control. C, protein bands of the immunoblot in B were quantified with ImageJ, and values were normalized to β-actin. Error bars are ± S.D. p-eIF2α, phosphorylation of the initiation factor eIF2 α. D, quantitative real-time PCR analysis of GRP78 and spliced XBP1 mRNA expression in DU145 cells treated with a pathological dose of anti-GRP78 AutoAbs (60 μg/ml). Results are expressed as -fold induction over non-treated (NT) cells (*, p < 0.05; n = 3). E, pathological doses of anti-GRP78 AutoAbs (60 μg/ml) do not increase GRP78 expression in PC-3 cells; thapsigargin (Tg; 300 nm) or tunicamycin (Tm; 2.5 μg/ml) was used as a control UPR inducer. β-Actin was used as a loading control.

Article Snippet: Membranes were blocked overnight in Tris-buffered saline plus 0.1% Tween 20 (TBST) containing 5% skim milk and subsequently incubated with the following primary antibodies: anti-GRP78/Bip (catalog no. 610979, BD Transduction), anti-phospho-eIF2α (catalog no. 9721S, BD Transduction), anti-VEGF pathway sampler kit (catalog no. 8696, Cell Signaling, Danvers, MA), anti-TF (catalog no. 4502, American Diagnostica, Stamford, CT), anti-CHOP (catalog no. B3, Santa Cruz Biotechnology, Inc., Dallas, TX) followed by the appropriate HRP-conjugated secondary antibodies (Dako, Carpentaria, CA) diluted in TBST containing 1% skim milk.

Techniques: Expressing, Flow Cytometry, Activation Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis

doi: 10.1371/journal.pone.0101164

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: P-eIF2α (S51) , Enzo Biosc. (Rb) , IF/WB , 1∶400/1∶2000.

Techniques:

Journal: Redox Biology

Article Title: Polysulfide and persulfide-mediated activation of the PERK-eIF2α-ATF4 pathway increases Sestrin2 expression and reduces methylglyoxal toxicity

doi: 10.1016/j.redox.2024.103450

Figure Lengend Snippet: Polysulfide-induced phosphorylation of eIF2α and nuclear accumulation of ATF4 are mediated by PERK phosphorylation in SH-SY5Y cells. (A) Cells were treated with 100 μM Na 2 S 2 for the indicated time, and levels of p-eIF2α and eIF2α in the cytoplasmic fractions were analyzed by western blotting. (B) Cells were treated with the indicated Na 2 S 2 concentrations for 2 h, and levels of p-eIF2α and eIF2α in cytosolic fractions were analyzed by western blotting. The p-eIF2α band densities were measured, and the ratio to eIF2α was calculated and expressed as the fold-change of the band levels relative to 0 h (A) or 0 μM (B) samples. (C) Cells were treated with 100 μM Na 2 S 2 for the indicated time, and ATF4 levels in the nuclear fractions were analyzed by western blotting. LaminB1 was used as a nuclear loading control (bottom panel). (D) Cells were treated with the indicated Na 2 S 2 concentrations for 2 h, and ATF4 levels in the nuclear fractions were analyzed by western blotting. LaminB1 was used as a nuclear loading control (bottom panel). The ATF4 band densities were measured, and the ratio to LaminB1 was calculated and expressed as the fold-change of the band levels relative to the 0 h (C) or 0 μM (D) samples. (E and F) SH-SY5Y cells transfected with siRNA were treated with 100 μM Na 2 S 2 for 0.5 h (for cytoplasmic proteins) or 2 h (for nuclear proteins) and lysed to prepare the cytoplasmic and nuclear fractions, respectively. Western blotting was performed on the cytoplasmic or nuclear proteins using anti-PERK (E) or anti-ATF4 antibodies, respectively (F). β-actin and LaminB1 were used as loading controls for cytoplasmic and nuclear proteins, respectively. Protein levels were quantified by densitometry, normalized to the loading control (β-actin or LaminB1), and expressed as a fold of the protein level detected in si Ctrl-transfected (scrambled) cells. Values indicate means ± S.D. Dunnett's test (A–D). Tukey's test (E and F). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. All data are representative of at least three independent experiments.

Article Snippet: The membranes were incubated with the following primary antibodies: ATF4 (10835-1-AP, Proteintech), CBS (ab96252, Abcam), eIF2α (GTX101241, Gene Tex), p-eIF2α (Ser51) (3398, Cell Signaling Technology), 3-MST (HPA001240, Sigma-Aldrich), Nrf2 (H-300; Santa Cruz Biotechnology, CA, USA), PERK (3192, Cell Signaling Technology), p-PERK (ab192591 Phospho T982, Abcam), PTP1B (5311, Cell Signaling Technology), Sestrin2 (10795-1-AP, Proteintech), β-actin (5125, Cell Signaling Technology), LaminB1 (12586, Cell Signaling Technology), and β-Tubulin (5346, Cell Signaling Technology).

Techniques: Western Blot, Control, Transfection

Journal: Journal of Lipid Research

Article Title: Curcumin inhibits lipolysis via suppression of ER stress in adipose tissue and prevents hepatic insulin resistance [S]

doi: 10.1194/jlr.M067397

Figure Lengend Snippet: Curcumin suppressed ER stress in adipose tissue. A: Expressions of p-IRE1α/IRE1α and p-eIF2α/eIF2α in adipose tissue exposed to PA were detected by Western blot. B: p-IRE1α/IRE1α and p-eIF2α/eIF2α expression in adipose tissue of HFD-fed mice. The results are expressed as the mean ± SD of four separate experiments. * P < 0.05 versus model; # P < 0.05 versus control.

Article Snippet: The antibodies of phospho-eukaryotic initiation factor 2α (eIF2α), eIF2α, PDE3B, goat anti-rabbit IgG (H+L) HRP, goat anti-rabbit IgG (H+L) HRP, GAPDH, Akt (A444), and Na + /K + -ATPase α1 (G19) were from Bioworld Technology (St. Paul, MN).

Techniques: Western Blot, Expressing

Journal: Journal of Lipid Research

Article Title: Curcumin inhibits lipolysis via suppression of ER stress in adipose tissue and prevents hepatic insulin resistance [S]

doi: 10.1194/jlr.M067397

Figure Lengend Snippet: AMPK played an important role in the inhibition of ER stress and lipolysis by curcumin. Adipose tissues were incubated with curcumin for 4 h. A: AMPK phosphorylation was determined by Western blot. B: Adipose tissue was incubated with curcumin followed by stimulation with PA for 24 h; IRE1α and eIF2α phosphorylation was determined by Western blot. C: The 3T3-L1 cells were transfected with AMPKα1/α2 or control siRNAs; expression of p-IRE1α and p-eIF2α was determined by Western blot. D: The p65 phosphorylation in adipose tissue exposed to thapsigargin (Thaps) stimulation for 24 h was detected by Western blot. E: PDE3B expression in adipocytes subjected to Thaps challenge when AMPKα1/α2 or control siRNAs were silenced with siRNAs. F: The 3T3-L1 cells were transfected with AMPKα1/α2 or control siRNAs, content of IL-6 in the medium was detected by ELISA kit (n = 6) and CM was used to incubate hepatocytes for 24 h. G: Insulin-mediated Akt phosphorylation in hepatocytes was detected by Western blot. The results are expressed as the mean ± SD of three independent experiments. * P < 0.05 versus model; # P < 0.05 versus indicated treatment.

Article Snippet: The antibodies of phospho-eukaryotic initiation factor 2α (eIF2α), eIF2α, PDE3B, goat anti-rabbit IgG (H+L) HRP, goat anti-rabbit IgG (H+L) HRP, GAPDH, Akt (A444), and Na + /K + -ATPase α1 (G19) were from Bioworld Technology (St. Paul, MN).

Techniques: Inhibition, Incubation, Western Blot, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Western blots of phospho-PKR ( A ) and -eIF2 α ( B ) in gastrocnemius muscle of mice bearing the MAC16 tumour after 5 days treatment with a PKR inhibitor (1 and 5 mg kg −1 ), as described in the legend to . The blots for total PKR and eIF2 α were used as loading controls. The first lane (CON) used gastrocnemius muscle from an NTB control. Representative blots are shown and the densitometric analysis gives the ratio of the phospho to total forms as an average of three separate blots ( n =9). Differences from NTB control are shown as a: P <0.05 or c: P <0.001, whereas differences from the solvent control are indicated as f: P <0.01.

Article Snippet: Rabbit monoclonal antibodies to phospho-eIF2 α (Ser 51) and to phospho-PKR (Thr 446) were purchased from Insight Biotechnology Ltd (London, UK).

Techniques: Western Blot, Control, Solvent

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Flow diagram showing how activation of PKR leads to inhibition of protein synthesis through phosphorylation of eIF2 α ; and increased protein degradation through activation of NF- κ B, which would be attenuated by a PKR inhibitor (PKRI).

Article Snippet: Rabbit monoclonal antibodies to phospho-eIF2 α (Ser 51) and to phospho-PKR (Thr 446) were purchased from Insight Biotechnology Ltd (London, UK).

Techniques: Activation Assay, Inhibition, Phospho-proteomics