Journal: Physiological Reports

Article Title: Mechanism of paraventricular nucleus H 2 S on PERK / TXNIP / NLRP3 pathway in male spontaneously hypertensive rats

doi: 10.14814/phy2.70861

Figure Lengend Snippet: Effects of SAMe and GSK on the expression of CBS, PERK/TXNIP/NLRP3 pathway‐related proteins, and inflammatory cytokines in the PVN of SHR. (a) Protein electropherograms of CBS, p‐PERK/PERK, TXNIP, NLRP3, ASC, pro‐Caspase‐1, Caspase‐1 p20, IL‐1β, TNF‐α, and IL‐18. (b–k) Results of statistical analysis of the related proteins and inflammatory factors in the PVN of each group of rats, respectively. All data are expressed as a mean ± SD. n = 6 in each group. SAMe (an endogenous H 2 S agonist), GSK (a PERK inhibitor).

Article Snippet: The membrane was sealed with BSA or skimmed milk powder for 1 h. Following the sealing, the membrane was exposed to the following primary antibodies at 4°C in the refrigerator overnight: anti‐β‐actin (1:1800; TA‐09; ZSGB‐BIO), CBS (1:1000; sc‐515180; Santa Cruz Biotechnology), PERK (1:2000; WL03378; WanLei), p‐PERK (1:2000; bs‐3330R; Bioss), TXNIP (1:1500; ab232330; Abcam), NLRP3 (1:2000; ab314905; Abcam), ASC (1:1000; AF6234; Beyotime Biotechnology ), pro‐Caspase‐1 (1:1500; sc‐56036; Santa Cruz Biotechnology), Caspase‐1 p20 (1:2000; WL02996a; WanLei), IL‐1β (1:1000; ab234437; Abcam), TNF‐α (1:1000; ab215188; Abcam), and IL‐18 (1:2000; AF5207; Beyotime Biotechnology ).

Techniques: Expressing

Journal: Physiological Reports

Article Title: Mechanism of paraventricular nucleus H 2 S on PERK / TXNIP / NLRP3 pathway in male spontaneously hypertensive rats

doi: 10.14814/phy2.70861

Figure Lengend Snippet: Effect of SAMe and GSK treatment on p‐PERK expression in the PVN of SHR. (a) Immunofluorescence staining of p‐PERK in the rat PVN, scale bar = 50 μm. (b) Statistical graph in average absorbance of p‐PERK in the rat PVN. All data are expressed as a mean ± SD. n = 4 in each group. SAMe (an endogenous H 2 S agonist), GSK (a PERK inhibitor).

Article Snippet: The membrane was sealed with BSA or skimmed milk powder for 1 h. Following the sealing, the membrane was exposed to the following primary antibodies at 4°C in the refrigerator overnight: anti‐β‐actin (1:1800; TA‐09; ZSGB‐BIO), CBS (1:1000; sc‐515180; Santa Cruz Biotechnology), PERK (1:2000; WL03378; WanLei), p‐PERK (1:2000; bs‐3330R; Bioss), TXNIP (1:1500; ab232330; Abcam), NLRP3 (1:2000; ab314905; Abcam), ASC (1:1000; AF6234; Beyotime Biotechnology ), pro‐Caspase‐1 (1:1500; sc‐56036; Santa Cruz Biotechnology), Caspase‐1 p20 (1:2000; WL02996a; WanLei), IL‐1β (1:1000; ab234437; Abcam), TNF‐α (1:1000; ab215188; Abcam), and IL‐18 (1:2000; AF5207; Beyotime Biotechnology ).

Techniques: Expressing, Immunofluorescence, Staining

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: 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: 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