Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet:

Article Snippet: IRE1 , OriGene , Cat# RC215023.

Techniques: Recombinant, SYBR Green Assay, Isolation, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: Strontium Attenuates Hippocampal Damage via Suppressing Neuroinflammation in High-Fat Diet-Induced NAFLD Mice

doi: 10.3390/ijms241210248

Figure Lengend Snippet: Sr restrained the HFD-induced apoptosis by altering expression levels of proteins related to the ERS pathway. ( A ) Western blot analysis of caspase-3, GRP78, IRE1α, p-IRE1α, XBP1, eIF2α, p-eIF2α, ATF4, ATF6, CHOP, and β-actin. ( B – K ) Relative protein expression of caspase-3 ( B ), GRP78 ( C ), IRE1α ( D ), p-IRE1α ( E ), XBP1 ( F ), eIF2α ( G ), p-eIF2α ( H ), ATF4 ( I ), ATF6 ( J ), and CHOP ( K ) in the hippocampi of each group of mice was examined through Western blotting ( n = 6 per group). Data were normalized with respect to the band of β-actin: the expression of target protein = the intensity of target protein band/the intensity of β-actin band. Results are shown as the ratio of the experimental group to the control group, and the values of the control group were taken as 1. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Subsequently, the following primary antibodies were used to incubate the membranes overnight at 4 °C: rabbit anti- NF-κB (#8242), rabbit anti- p38 (#9212), rabbit anti- ERK (#9102), rabbit anti-phospho- ERK ( p-ERK , #4370), rabbit anti-phospho- p38 ( p-p38 , #4511), and anti- caspase-3 (#9662) (purchased from Cell Signaling Technology (Danvers, MA, USA)); mouse anti- ATF6 (EM1701-94) (purchased from Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China); rabbit anti- XBP1 (A1731), rabbit anti-phospho- NF-κB ( p- NF-κB , AP0475), rabbit anti- GRP78 (A0241), and mouse anti- β-actin (purchased from Wuhan ABclonal Technology Co., Ltd., Wuhan, China); rabbit anti- eIF2α (ab115822), rabbit anti- TLR4 (ab13556), and rabbit anti-phospho- eIF2α ( p-eIF2α , ab32157) (purchased from Abcam (Cambridge, MA, USA)); and rabbit anti- CHOP (BM4962), anti-phospho- IRE1α ( p-IRE1α , BM4444), rabbit anti- ATF4 (BM5179), and rabbit anti- IRE1α (A00683-1) (purchased from Wuhan BOSTER Biological Technology Co., Ltd., Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects against Aβ toxicity via attenuating Aβ-induced endoplasmic reticulum stress

doi: 10.1186/s12974-019-1429-0

Figure Lengend Snippet: Recombination human MANF protein (rhMANF) protects against Aβ 1–42 -induced cell toxicity. a The dose-dependent effect of rhMANF on cell viability in the presence of Aβ 1–42 exposure. SH-SY5Y cells were pretreated with different concentrations of rhMANF protein (0.5, 1, and 2.0 mg/ml) for 4 h prior to the Aβ 1–42 (10 μM) treatment for additional 24 h and processed for the MTT assay. * P < 0.05, compared with the cells only treated with Aβ 1–42 . b The time-course of rhMANF on cell viability in the presence of Aβ 1–42 . SH-SY5Y were pretreated with rhMANF (2.0 mg/ml) for 4 h following the Aβ 1–42 (10 μM) treatment for indicated times and processed for the MTT assay. * P < 0.05, compared with the only Aβ 1–42 -treated cells at indicated time points. c The protein levels of BiP, ATF6, phospho-IRE1, XBP1s, phospho-eIF2α, ATF4, CHOP, and cleaved caspase-3 were determined in SH-SY5Y cells treated with Aβ 1–42 (10 μM) for 24 h with or without rhMANF. GAPDH was used as a loading control. d The densitometric quantitation of indicated proteins normalized to GAPDH levels in c. * P < 0.05, ** P < 0.01, compared with the cells only treated with Aβ 1–42 . # P < 0.05, ## P < 0.01, ### P < 0.001, compared with control group. All the quantitative data were presented as mean + SD of at least three independent experiments. C - caspase - 3 cleaved caspase-3

Article Snippet: The proteins were transferred to PVDF membranes and blocked in 5% nonfat milk at room temperature for 1 h, then incubated at 4 °C overnight with the following primary antibodies: rabbit anti-MANF antibody (1: 1000, Abcam, ab67271), rabbit anti-BiP antibody (1:1000, proteintech, 11587-1-ap), rabbit anti-CHOP antibody (1:1000, proteintech, 15204-1-AP), rabbit anti-phospho-eIF2α antibody (1:1000, CST, 3398 s), rabbit anti-phospho-IRE1 antibody (1:1000, Bioss, bs-4308R), rabbit anti-XBP1s antibody (1:1000, Biolegend, 619502), mouse anti-ATF4 antibody (1:1000, CST, 11815 s), rabbit anti-ATF6 antibody (1:1000, Proteintech, 24169-1-AP), rabbit anti-cleaved-caspase 3 antibody (1: 1000, CST, 9664S), mouse anti-α-tubulin (1: 1000, sigma, t6199), and rabbit-anti-GAPDH (1: 1000, Elabscience, E-AB-20059).

Techniques: MTT Assay, Quantitation Assay

Journal: Journal of Neuroinflammation

Article Title: Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects against Aβ toxicity via attenuating Aβ-induced endoplasmic reticulum stress

doi: 10.1186/s12974-019-1429-0

Figure Lengend Snippet: MANF protects against Aβ 1–42 toxicity via inhibiting ER stress. a Effect of MANF overexpression on the levels of BiP, ATF6, phospho-IRE1, XBP1s, phospho-eIF2α, ATF4, CHOP, and cleaved caspase-3 in Aβ 1–42 -treated cells. N2a cells were transfected with pcDNA3.1-MANF-Flag plasmid and its empty vector before Aβ 1–42 (10 μM) treatment for 24 h or TM (2.5 μg/ml) treatment for 12 h and processed for WB. b Effect of MANF knockdown on the levels of BiP, ATF6, phospho-IRE1, XBP1s, phospho-eIF2α, ATF4, CHOP, and cleaved caspase-3 in Aβ 1–42 -treated cells. N2a cells were transfected with NC-siRNA or MANF-siRNA before Aβ 1–42 (10 μM) treatment for 24 h or TM (2.5 μg/ml) treatment for 12 h, respectively, then processed for WB. c Quantitation of proteins normalized to α-tubulin levels in a. d Quantitation of proteins normalized to α-tubulin levels in b . All the quantitative data were presented as mean ± SD of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.01 . TM tunicamycin, C - caspase - 3 cleaved caspase-3

Article Snippet: The proteins were transferred to PVDF membranes and blocked in 5% nonfat milk at room temperature for 1 h, then incubated at 4 °C overnight with the following primary antibodies: rabbit anti-MANF antibody (1: 1000, Abcam, ab67271), rabbit anti-BiP antibody (1:1000, proteintech, 11587-1-ap), rabbit anti-CHOP antibody (1:1000, proteintech, 15204-1-AP), rabbit anti-phospho-eIF2α antibody (1:1000, CST, 3398 s), rabbit anti-phospho-IRE1 antibody (1:1000, Bioss, bs-4308R), rabbit anti-XBP1s antibody (1:1000, Biolegend, 619502), mouse anti-ATF4 antibody (1:1000, CST, 11815 s), rabbit anti-ATF6 antibody (1:1000, Proteintech, 24169-1-AP), rabbit anti-cleaved-caspase 3 antibody (1: 1000, CST, 9664S), mouse anti-α-tubulin (1: 1000, sigma, t6199), and rabbit-anti-GAPDH (1: 1000, Elabscience, E-AB-20059).

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitation Assay

Journal: Journal of Lipid Research

Article Title: ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

doi: 10.1016/j.jlr.2022.100279

Figure Lengend Snippet: ER stress induces UPR mt in an IRE1 kinase-dependent manner. A–E: Wild type (WT) MEFs were treated with KIRA6 (5 μM) and TG (100 nM) for 12 h, and total RNA was analyzed by qRT-PCR for Atf5, Atf4, Ddit3, Lonp1, Hspa9, and Gapdh mRNA (n = 4 biological replicates). F: WT MEFs were treated with KIRA6 (5 μM) and TG (100 nM) for 4 h, and MF and CE protein lysates were analyzed by Western blotting using specific antibodies against LONP1, mtHSP70, and CLPP (for MF) and p-IRE1, total IRE1, and β-actin (for CE). The membrane was stained by Ponceau S (n = 3 biological replicates; a representative blot is shown). G–K: WT MEFs were treated with 2 μM AMG18 and 100 nM TG for 12 h, and total RNA was analyzed by qRT-PCR for Atf5, Atf4, Ddit3, Lonp1, Hspa9, and Gapdh mRNA (n = 4 biological replicates). Data information: All data are mean ± SEM; (n = 4). Unpaired t test with Welch’s correction. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤0.001, ∗∗∗∗ P ≤0.0001. ATF, activating transcription factor; CE, total cellular extract; CLPP1, caseinolytic protease-1; ER, endoplasmic reticulum; IRE1, inositol-requiring enzyme-1; MEFs, mouse embryonic fibroblasts; MF, mitochondrial fraction; mt, mitochondria; mtHSP70, mitochondrial heat shock protein 70; LONP1, Lon protease-1; TG, thapsigargin; UPR, unfolded protein response.

Article Snippet: IRE1 kinase inhibitor, KIRA6 (19151), SPL inhibitor [2-acetyl-5-tetrahydroxybutyl imidazole, THI (13222)], and SPL Fluorogenic Substrate (13238) were purchased from Cayman.

Techniques: Quantitative RT-PCR, Western Blot, Membrane, Staining

Journal: Journal of Lipid Research

Article Title: ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

doi: 10.1016/j.jlr.2022.100279

Figure Lengend Snippet: ER stress suppresses SPL activity and induces cellular S1P levels. A: S1P degradation by SPL activity. B–E: SPL fluorogenic substrate assay was performed according to the manufacturer protocol on the cellular lysates from: (B) HEK293 cells that were treated with KIRA6 (20 μM) or 4μ8c (100 μM) and TG (600 nM; 2 h) (n = 6 biological replicates); C: HEK293 cells were that were treated with AMG18 (5–10 μM) and TG (600 nM; 2 h) (n = 6 biological replicates); and D–E: HEK293 cells that were transfected with the indicated plasmids and treated with TG (600 nM; 2 h) (n = 4 biological replicates for D and n = 6 biological replicates for E). F–I: lipid quantification was performed by LC-MS/MS from lysates of HEK293 that were (F–G) pretreated with KIRA6 (20 μM; 1 h) and then treated with TG (600 nM) or vehicle for 2 h (n = 4 biological replicates for DMSO, KIRA6, and TG, n = 3 biological replicates for KIRA6+TG), S1P is measured from total cell lysates (F) and from isolated mitochondria (G, H) transfected with scrambled (siSCR) or IRE1-specific (siIRE1) siRNA (50 nM) and treated with TG (600 nM; 1 h) (n = 5 biological replicates), or (I) pretreated with AMG18 (5 μM; 1 h), followed by TG (600 nM; 1 h) treatment (n = 3 biological replicates for TG and n = 5 biological replicates for AMG18+TG). Peak areas of S1P were sequentially normalized by the internal standard and protein concentrations. Higher fluorogenic signal indicates higher SPL enzymatic activity in A–E. Data information: B–F data are mean ± SEM; unpaired t test with Welch’s correction. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤0.001, ∗∗∗∗ P ≤0.0001, G–H are mean ± SEM; unpaired t test. ∗ P ≤ 0.05. ER, endoplasmic reticulum; HEK293, Human embryonic kidney 293; IRE1, inositol-requiring enzyme-1; S1P, sphingosine 1-phosphate; SPL, sphingosine 1-phosphate lyase; TG, thapsigargin.

Article Snippet: IRE1 kinase inhibitor, KIRA6 (19151), SPL inhibitor [2-acetyl-5-tetrahydroxybutyl imidazole, THI (13222)], and SPL Fluorogenic Substrate (13238) were purchased from Cayman.

Techniques: Activity Assay, Transfection, Liquid Chromatography with Mass Spectroscopy, Isolation