Journal: Inflammation

Article Title: Scutellarin Alleviates Ovalbumin-Induced Airway Remodeling in Mice and TGF-β-Induced Pro-fibrotic Phenotype in Human Bronchial Epithelial Cells via MAPK and Smad2/3 Signaling Pathways

doi: 10.1007/s10753-023-01947-7

Figure Lengend Snippet: Scutellarin inactivates the Smad and MAPK pathways in TGF-β1-treated 16HBE cells. a , b The protein levels of phosphorylated Smad2/Smad3 and total Smad4 were evaluated by western blotting. c , d The effect of scutellarin on the formation of Smad2/3/4 complex in TGF-β1-treated 16HBE cells was examined by co-immunoprecipitation. e , f The protein levels of phosphorylated ERK, phosphorylated JNK, and phosphorylated p38 were measured by western blotting. Data are analyzed by one-way analysis of variance followed by Tukey’s post hoc analysis and expressed as the mean ± standard deviation of three independent experiments. ** p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the TGF-β1 group.

Article Snippet: After blocking using 5% skimmed milk for 2 h, the membranes were incubated overnight with primary antibodies against phosphorylated Smad2 (ab280888, 1:1000; Abcam, Shanghai, China), phosphorylated Smad3 (ab52903, 1:2000; Abcam), Smad2/3 (ab202445, 1:1000; Abcam), β-actin (ab6276, 1:5000; Abcam), phosphorylated ERK (sc-135760, 1:1000; Santa Cruz Biotechnology, Shanghai, China), Smad4 (ab40759, 1:5000; Abcam), ERK (sc-398015, 1:500; Santa Cruz Biotechnology), p38 (ab170099, 1:2500; Abcam), phosphorylated JNK (sc-6254, 1:1000; Santa Cruz Biotechnology), JNK (sc-7345, 1:250; Santa Cruz Biotechnology), phosphorylated p38 (ab195049, 1:1000; Abcam), E-cadherin (sc-8426, 1:1000; Santa Cruz Biotechnology), α-SMA (ab5694, 1:500; Abcam), and N-cadherin (ab76011, 1:5000; Abcam) at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Standard Deviation, Control

Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Western blot analysis results for ERS-associated proteins. (A) The representative blotting images and (B) quantified data demonstrate elevated p-PERK, eIF2a, CHOP, Bax/Bcl-2, Caspase-12 and cleaved Caspase-3 level in L539fs/47-hERG cells, which can be reversed by 4-PBA. Each experiment was repeated at least three times. # P<0.05 between the three model cell groups; * P<0.05 between two subgroups treated with or without 4-PBA intervention. P<0.05 was considered to indicate a statistically significant difference. ERS, endoplasmic reticulum stress; hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Proposed mechanisms of mutated hERG-induced cell apoptosis. Mutated L539fs/47-hERG protein retained in the ER lumen provokes ERS. Elevated expression levels of GRP78 activate the PERK-eIF2α-CHOP pathway, which is important in the mechanism of L539fs/47-hERG-induced ERS-mediated cell apoptosis. ER-specific caspase-12 is involved in the apoptotic mechanisms caused by the hERG mutation through cleaving and activating caspase-3. The pair of apoptosis factors Bax/Bcl-2 also regulate L539fs/47-hERG-induced cell apoptosis. hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; ER, endoplasmic reticulum; ERS, ER stress; ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis

Journal: Neurobiology of disease

Article Title: Calcineurin β protects brain after injury by activating the unfolded protein response

doi: 10.1016/j.nbd.2016.06.011

Figure Lengend Snippet: CNβ physically interacts with PERK and promotes PERK phosphorylation and oligomerization. (A) C8D1A type I astrocytes were treated with vehicle (DMSO; Con) or 1 µM of thapsigargin (Tg) for 1 h. Cell lysates were analyzed for total (T) and phosphorylated (P) PERK and eIF2α using immunoblots. (B) Densitometry histograms after normalization to T-PERK or T-eIF2α, respectively (n = 3, mean ± SEM, ** p < 0.01 and *** p < 0.001 by unpaired two-tailed Student’s t -test). (C) Primary mouse astrocytes were treated as indicated in (A) and then cross-linked with disuccinimidyl suberate (DSS) for 30 min. Immunoprecipitation (IP) was performed with anti-CNβ antibody and subsequent blots were probed with anti-PERK antibody. (D) Quantification of P-PERK/T-PERK densitometric ratio in (C) (n = 3, mean ± SEM, * p < 0.05 by unpaired two-tailed Student’s t test). (E) GST pull-down assay with either 8 nM of CNα or CNβ. The proteins were incubated with glutathione sepharose 4B for 1 h, resolved on a 12% SDS-polyacrylamide gel and probed with an anti-calcineurin PAN-A antibody. CN pull-down levels are shown for GST alone and GST-cPERK. (F) Densitometric histogram of (E) (n = 3, mean ± SEM, ** p < 0.01 by unpaired two-tailed Student’s t test). (G) GST-cPERK was added to all reaction mixtures along with [g 32 P] ATP in the absence or the presence of either of 0.043 mM of CNα or CNβ. Reaction mixtures were run on SDS-PAGE and visualized by autoradiography. (H) Quantification of cPERK auto-phosphorylation density (ne = 5, mean ± SEM, * p < 0.05 by one-way ANOVA). (I) Recombinant His-CNβ and GST-cPERK were incubated at the concentrations indicated, in the presence of 0.3 mM of DSS cross-linker for 30 min at room temperature. The ensuing protein complexes were run on SDS-PAGE and detected by immunoblotting using an anti-PERK antibody. (J) Recombinant His-CNβ (1.2 mM) and GST-cPERK (0.01 mM) were incubated in the presence of 0.3 mM of DSS for 30 min at room temperature. The protein complexes were run on SDS-PAGE and detected by immunoblotting using antibodies against CNβ and PERK.

Article Snippet: Primary antibodies used were rabbit anti-phosphorylated PERK (conjugated to Alexa 647, #bs-3330R–A647; Bioss), rabbit anti-CNα (#13422-1-AP; Proteintech), rabbit anti-CNβ (#07–1439; Millipore), rabbit anti-phosphorylated eIF2α (P-eIF2α, #µA5 – 15133; Thermo scientific) and mouse anti-GFAP (#MAB 360; Millipore).

Techniques: Western Blot, Two Tailed Test, Immunoprecipitation, Pull Down Assay, Incubation, SDS Page, Autoradiography, Recombinant