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

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: Biomedicines

Article Title: Inhibition of PERK Kinase, an Orchestrator of the Unfolded Protein Response (UPR), Significantly Reduces Apoptosis and Inflammation of Lung Epithelial Cells Triggered by SARS-CoV-2 ORF3a Protein

doi: 10.3390/biomedicines11061585

Figure Lengend Snippet: Transient transfection of A549 cells with ORF3a-mRNA. ( a ) Evaluation of ORF3a mRNA levels at 48, 72 and 96 h post-transfection by real-time PCR. ORF3a mRNA levels were normalized to GAPDH gene. ( b ) Detection of recombinant ORF3a protein levels 48, 72 and 96 h post-transfection by Western blotting. For the detection of recombinant ORF3a, a primary polyclonal anti-His tag antibody was used. ORF3a protein expression normalized to GAPDH protein levels. ( c ) Detection of Green Fluorescent Protein (GFP) protein levels 48, 72 and 96 h post-transfection by Western blotting. For the detection of GFP, a primary polyclonal anti-GFP antibody was used. GFP protein expression normalized to GAPDH protein levels. The notation (***) indicate statistically significant differences ( p ≤ 0.001) compared to control cells.

Article Snippet: PERK polyclonal rabbit primary antibody was obtained from OriGene (OriGene, Rockville, MD, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Recombinant, Western Blot, Expressing, Control

Journal: Experimental Animals

Article Title: Hairless-knockout piglets generated using the clustered regularly interspaced short palindromic repeat/CRISPR-associated-9 exhibit abnormalities in the skin and thymus

doi: 10.1538/expanim.19-0018

Figure Lengend Snippet: Western blot analysis of hairless (HR) in eyelid skin samples of HR −/− and WT piglets. HR was detected using a polyclonal anti-Hr antibody. A polyclonal anti-actin antibody was used as a loading control.

Article Snippet: Membranes were blocked with 5% skimmed milk and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-HR (1:1,000; Eterlife, Birmingham, UK) and goat polyclonal anti-actin (1:1,000; Santa CruzBiotechnology, Redwood, CA, USA).

Techniques: Western Blot, Control

Journal: BMC Biology

Article Title: The chromosome-level genome and key genes associated with mud-dwelling behavior and adaptations of hypoxia and noxious environments in loach ( Misgurnus anguillicaudatus )

doi: 10.1186/s12915-023-01517-1

Figure Lengend Snippet: Transcriptome analysis of the drug stress trial and the expression and location of fmo5 in loach Misgurnus anguillicaudatus . a The heatmap of several DGEs (namely, fmo genes, ugt genes, and endoplasmic reticulum stress (RE stress) genes (in red color)) between the control and drug stress group. b The expression profiles of fmo5 genes in livers of wild-type (WT) loach under five drug stress at different time points. c Expression and location of fmo5 gene in the loach. The black dotted box represents the expression signal (liver and intestine tissues) of fmo5 in the loach (top). Immunofluorescence (lower left; liver tissue) and immunohistochemistry (lower right; intestine tissue) of fmo5 in the loach. White and black arrows present the expression signal. H, hepatocyte; GC, goblet cell. Atf6 , cyclic AMP-dependent transcription factor 6; ugt1a1 , UDP-glucuronosyltransferase 1-1; eif2ak3 , eukaryotic translation initiation factor 2-alpha kinase 3; chop , DNA damage-inducible transcript 3 protein; atf4 , cyclic AMP-dependent transcription factor 4; Mis0185950.1, Mis0185970.1, Mis0185930.1 ( fmo5 ), dimethylaniline monooxygenase [N-oxide-forming] 5; Mis0072610, Mis0115360.1 ( ugt2a1 ), UDP-glucuronosyltransferase 2A1

Article Snippet: The polyclonal antibodies of Atf6 (Rabbit, 1:1000, A0202, ABclonal, China), Eif2ak3 (Rabbit, 1:1200, A18196, ABclonal, China), Muc2 (Rabbit, 1:1000, A14659, ABclonal, China), and Chop (produced by our laboratory) were diluted 50 times with the primary antibody dilution buffer (Gbcbio Technologies Inc., China).

Techniques: Expressing, Control, Immunofluorescence, Immunohistochemistry

Journal: BMC Biology

Article Title: The chromosome-level genome and key genes associated with mud-dwelling behavior and adaptations of hypoxia and noxious environments in loach ( Misgurnus anguillicaudatus )

doi: 10.1186/s12915-023-01517-1

Figure Lengend Snippet: Expansion in fmo5 (ID: Mis0185930.1) gene involved in the detoxification function of loach Misgurnus anguillicaudatus . a The growth curves of WT and fmo5 deletion ( fmo5 −/− ) loach. b–d Histological structure analysis of intestines from WT and fmo5 −/− loach. GC, goblet cell; FMH, mucosal fold height. e,f Expressions of muc2 and some endoplasmic reticulum stress (RE stress)-related genes in intestines. g Histological structures of livers from WT and fmo5 −/− loach. The black dotted boxes represent the injury area. The black and white triangles represent hepatocyte (H for short). The yellow triangles represent red blood cells (RBC for short). h,i Expressions of some RE stress-related genes in livers. muc2 , Mucin; atf6 , Cyclic AMP-dependent transcription factor; eif2ak3 , eukaryotic translation initiation factor 2-alpha kinase 3; chop , DNA damage-inducible transcript 3 protein; Gapdh, glyceraldehyde-3-phosphate dehydrogenase. Dapi, 4',6-diamidino-2-phenylindole. * significant difference ( p < 0.05); ** very significant difference ( p < 0.01); *** extremely significant difference ( p < 0.001)

Article Snippet: The polyclonal antibodies of Atf6 (Rabbit, 1:1000, A0202, ABclonal, China), Eif2ak3 (Rabbit, 1:1200, A18196, ABclonal, China), Muc2 (Rabbit, 1:1000, A14659, ABclonal, China), and Chop (produced by our laboratory) were diluted 50 times with the primary antibody dilution buffer (Gbcbio Technologies Inc., China).

Techniques: