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: Stem cells international

Article Title: Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1 α Branch of the Endoplasmic Reticulum Stress Response.

doi: 10.1155/2023/4483776

Figure Lengend Snippet: FIGURE 6: MSCs attenuated EMT via the IRE1α/XBP1 pathway. (a) The protein expression levels of IRE1α and p-IRE1α were measured using western blotting and quantified using densitometry in ImageJ software (n = 4, one-way ANOVA with Duncan’s post hoc test). (b) The mRNA expression level of IRE1α was measured using Q-PCR (n = 3, one-way ANOVA with Duncan’s post hoc test). (c) A549 cells were treated with 4 μ8c (5 and 10 μM) for 48 hr, and the protein expression levels of IRE1α and XBP-1s were measured using western blotting and quantified using densitometry in ImageJ software (n = 4, one-way ANOVA with Duncan’s post hoc test). (d) A549 cells were treated with 10 ng/ml TGF- β1 in the presence or absence of 4 μ8c for 72 hr. The protein expression levels of p-IRE1α, XBP-1s, E-cadherin and vimentin were measured using western blotting and quantified using densitometry in ImageJ software (n = 4, one-way ANOVA with Duncan’s post hoc test). The data are shown as the means Æ SEMs (∗∗∗P<0:001, ∗∗P<0:01, ∗P<0:05 vs. the control group; ###P<0:001, ##P<0:01, #P<0:05 vs. the TGF-β1 group).

Article Snippet: After the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to membranes, which were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1α (A00683-1, Boster, Wuhan, China), phospho-IRE1α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET161039, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight.

Techniques: Expressing, Western Blot, Software, Control

Journal: Stem cells international

Article Title: Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1 α Branch of the Endoplasmic Reticulum Stress Response.

doi: 10.1155/2023/4483776

Figure Lengend Snippet: FIGURE 9: MSCs attenuated ER stress and EMT in the lungs of mice with lung fibrosis. (a) The mRNA expression levels of E-cadherin and Vimentin in lung tissues were measured using Q-PCR (n = 5, one-way ANOVA with Duncan’s post hoc test). (b) The protein expression levels of E-cadherin and vimentin in lung tissues were measured using western blotting, and the results were quantified via densitometry by using ImageJ software (n = 3, one-way ANOVA with Duncan’s post hoc test). (c) The mRNA expression levels of Bip, Atf6, Atf4, Xbp-1s, Ire1α and Chop in lung tissues were measured using Q-PCR (n = 5, one-way ANOVA with Duncan’s post hoc test). (d) The protein expression levels of ATF6, ATF4, IRE1α, p-IRE1α, XBP-1s, XBP-1u, BiP and CHOP in lung tissues were measured using western blotting, and the results were quantified via densitometry by using ImageJ software (n = 3, one-way ANOVA with Duncan’s post hoc test). (e) Images of immunofluorescence staining of BiP (green) and vimentin (red) in the lung tissues of mice. Scale bar, 100 μm. The data are shown as the means Æ SEMs (∗∗P<0:01, ∗P<0:05 vs. the control group; #P<0:05, ##P<0:01 vs. the BLM group).

Article Snippet: After the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to membranes, which were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1α (A00683-1, Boster, Wuhan, China), phospho-IRE1α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET161039, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight.

Techniques: Expressing, Western Blot, Software, Staining, Control