Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Enhanced large conductance K + channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

doi: 10.1152/ajpheart.00636.2013

Figure Lengend Snippet: Comparison of BK-α- and BK-β-subunit expression in cerebral vessel homogenates isolated from FHH and FHH.1BN rats. BK-α- and BK-β-subunits were probed on different blots. The same blots were then reprobed for β-actin. A: representative bands corresponding to the molecular weight of the BK-α-subunit (∼100 kDa), BK-β-subunit (∼25 kDa), and β-actin (∼42 kDa) in cerebral vessels obtained from FHH and FHH.1BN rats (n = 8 animals each; 3 sets of experiments). B: a comparison of the expression of α- and β-subunits. Numbers in parentheses indicate the number of samples studied.

Article Snippet: After transfer, the membrane was blocked with TBS-T buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween, and a 5% blocking powder (Bio-Rad) at 4°C for 1 h. The blot was probed with primary antibody against BK α- and β-subunit [1:500 and 1:200, respectively; polyclonal rabbit Anti-K Ca 1.1, to amino acids 1184–1200 and Anti-sloβ1 (KCNMB1); Alomone Labs, Jerusalem, Israel] overnight at 4°C.

Techniques: Expressing, Isolation, Molecular Weight

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Protein expression levels of Cav1.2, Piezo1, CaM, and Src in human LAA tissues. (A) Representative western blots and densitometric analysis of Cav1.2 and Piezo1 proteins in LA tissues of AF patients and those with SR. (B) Representative western blots and densitometric analysis of CaM and Src protein in LA tissues of AF patients and those with SR. GAPDH was the internal control. ** p < 0.01. Values are presented as the mean ± standard error of the mean (SEM).

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effects of hypertension on the incidence of AF, I Ca,L , and Piezo1 expression in Wistar rats and SHRs with and without Val treatment. (A) Representative baseline surface ECG and intra-atrial electrocardiogram (IAEG); (B) The incidence AF in Wistar rats and SHRs treated with and without Val treatment ( n = 8). ** p < 0.01 vs. Wistar rat; ## p < < 0.01 vs. SHRs. (C) Typical surface ECG recordings of rats with AF that spontaneously reverted to SR and typical disorganized amplification of atrial waves (f wave). (D) Representative traces of AP in atrial myocytes from Wistar rats, SHRs, and SHR + Val groups and a histogram of APD in atrial myocytes from each group ( n = 12–15 myocytes from 3–4 rats). * p < 0.05 vs. Wistar rat; # p < 0.05 vs. SHRs. (E) Representative traces of I Ca,L (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in atrial myocytes of each group ( n = 8–14 myocytes from 3–4 rats). (F) Representative examples of immunohistochemical analysis of LA tissues from Wistar rats and SHRs treated with and without Val using Ab against Piezo1. Scale bar, 20 μm. (G) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in LA tissues of Wistar rats and SHRs. GAPDH was the internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Amplification, Activation Assay, Immunohistochemical staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of HHP on the depression of I Ca,L in HL-1 cells. (A) Representative traces of AP in HL-1 cells under various hydrostatic pressures (0, 20, and 40 mmHg) for 24 h. APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 10, and 7 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (B) Representative traces (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L ( n = 8–18 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (C) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in HL-1 cells under various hydrostatic pressure (0, 20, and 40 mmHg) for 24 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: The effects of Piezo1 on perceiving HHP and mediating the decrease of I Ca,L . (A,B) Representative Ca 2+ traces and Δ Ca i 2 + (ΔF/F) are shown. Ca 2+ entry was evoked by 10 μm Yoda1 in HL-1 cells stimulated by HHP in the presence or absence of the Piezo1 inhibitor GsmTx4 ( n = 50) or siRNA specifically knockdown Piezo1 ( n = 53–58). si-C, scrambled (control) siRNA; si-P, siRNA directed against Piezo1. (C) Representative traces of AP in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment (3.0 μM) and APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 7, and 11 at 0, 40, and 40 mmHg + GsmTx4). * p < 0.05, ** p < 0.01 vs. 0 mmHg; # p < 0.05 vs. 40 mmHg. (D) Representative traces (pulse protocol, inset), corresponding current–voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment ( n = 9–15). ** p < 0.01 vs. 0 mmHg; ## p < 0.01 vs. 40 mmHg. (E) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (F) Representative blots and densitometry analysis of Cav1.2 in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of CaM/Src on the decrease of I Ca,L induced by HHP or Yoda1 stimulation. (A) Representative blots and densitometry analysis of CaM and Src in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (B) Representative blots and densitometry analysis of CaM and Src in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. (C) Representative blots and densitometry analysis of Src and p-Src ( n = 4) in HL-1 cells transfected with scrambled (control) siRNA or siRNA directed against Piezo1 for 48 h, then treated with Yoda1 at different dosages (0, 1, and 3 μM) for 15 min. (D) Representative traces of AP and histogram of APD in HL-1 cells ( n = 7–8). * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (E) Current–voltage relationship for I Ca,L ( n = 9–17) in HL-1 cells stimulated by 40 mmHg pressure treated with 15 μM PP1 or W7. * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (F) Representative blots and densitometry analysis of Cav1.2 and Src in HL-1 cells stimulated by 40 mmHg pressure treated with W7 under different concentrations (5, 10, 15, and 20 μM). (G) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure treated with PP1 (15 μM). (H) Current-voltage relationship for I Ca,L ( n = 8–10) in HL-1 cells stimulated by Yoda1(3 μM) treated with PP1. * p < 0.05, ** p < 0.01 vs. DMSO. # p < 0.05, ## p < 0.01 vs. Yoda1. (I) Representative blots and densitometry analysis of Cav1.2 in Yoda1(3μM) -stimulated HL-1 cells treated with PP1 (15 μM). GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Schematic representation of the mechanism for the decrease of I Ca,L induced by HHP. Piezo1 activated by HHP depressed I Ca,L contributing to increased AF susceptibility through the CaM/Src/Pitx2 pathway.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques:

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. qPCR results from pig articular chondrocytes stimulated with OSM and treated with or without SRC inhibitor (SU6656) for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Protein expression of pSRC in ATDC5 cells after transfection (72 hours) with the modified and WT gp130 plasmids stimulated with OSM for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. C. qPCR results from ATDC5 cells treated with SRC inhibitor (SU6656) alone, or transfected with indicated variants of plasmids and stimulated with or without OSM for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. D. Protein expression of gp130 pY814 in untreated fetal (17 weeks) and adult articular chondrocytes. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Transfection, Modification

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Protein expression of pSRC in pig articular chondrocytes stimulated with IL-6 cytokines. Horizontal lines with bars show the mean ± SD. n=3. B. Schematic of modified amino acids within gp130 812-827 domain. Protein expression of pSRC in Ba/F3 cells after transfection with the modified or WT gp130 plasmids stimulated with IL-6 for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. C. CRISPR gRNA was designed by PNA Bio. Y814 mutant mouse (F814) was generated by the USC transgenic mouse core. Mice genotyping was performed by GeneWiz. Representative images of 4-month-old males shown. n=4. D. Protein expression of gp130 pY814 in wild type (WT) and F814 mouse splenocytes treated with and without OSM and LIF for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. E. Distribution of differentially expressed genes in MA plot of wild type (WT) and F814 mouse periarticular stromal cells. MA plots (log2fold change vs log2mean expression) for WT-OSM vs WT and Y814-OSM vs Y814 mouse periarticular stromal cells. Each dot represents a gene. Red dots represent upregulated genes i.e. log2fold-change >1 and p-value<0.05. Blue dots represent downregulated genes i.e. log2fold change < −1 and p-value 0.05. Genes that do not qualify this threshold are indicated by grey dots. F. Levels of protein complex formation between gp130 and pSRC in wild type and F814 mouse splenocytes stimulated with or without OSM for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. G. Protein expression of pSRC in wild type and F814 mouse splenocytes stimulated with or without OSM for 24 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Modification, Transfection, CRISPR, Mutagenesis, Generated, Transgenic Assay

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Protein expression of gp130 pY814 in wild type (WT) and F814 mouse splenocytes treated with and without OSM and LIF for 4 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Levels of complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Protein levels of pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. C. Computational analysis using GOLD software predicted a high affinity binding of peptide QQpYF to the regulatory site of SRC (c-SRC). c-SRC as visualized by crystallography. The structure of the indicated c-SRC domains is shown in ribbon diagram representation (left) as well as with electrostatic potential (blue, positive charge; red, negative charge; white, neutral) mapped onto the molecular surface (right). Peptide QQpYF is shown in stick representation. D. Transcription of genes was determined via qPCR in human adult OA articular chondrocytes treated with or without peptide QQpYF for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. E. Pig knee cartilage explants were stimulated with or without OSM and treated with or without peptide QQpYF at indicated doses for 72 hours followed by a neoepitope assay. Levels of cleaved ACAN and COL2 neoepitopes in the supernatant were quantified with respect to the wet weight of the explant. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Software, Binding Assay

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Levels of complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 4 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Protein levels of pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 4 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques:

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Protein expression of gp130 pY814 in pig articular chondrocytes treated with or without OSM and R805 for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Gross morphology and Masson’s trichrome staining of bearded dragon treated with vehicle control and R805 14 days post-amputation. Dashed lines mark amputation planes. n=10. sc=spinal cord; we=wound epithelium; ve=vertebra. C. Gross morphology of bearded dragon co-treated with vehicle control or R805 and PBS or clodronate liposomes 14 days post-amputation. Dashed lines mark amputation planes. n=10. D. Mouse vehicle-treated or R805-treated post-wound day (PWD) 21 wound sections. Representative images are shown. n=8. E. Hair follicle (n=3) and fiber length (n=8) in R805-treated mouse PWD 14 and untreated control wounds. Horizontal lines with bars show the mean ± SD. F. Re-clustering of clusters annotated as macrophages from skin wounds of vehicle-treated or R805-treated mice PWD 14. Dot plots depict gene expression in each macrophage cluster. The contribution of each sample to each cluster is shown as a stacked bar graph. Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. DCs = dendritic cells. G. Re-clustering of clusters annotated as fibroblasts from skin wounds of vehicle-treated or R805-treated mice PWD 14. Dot plots depict gene expression in each fibroblast cluster. The contribution of each sample to each cluster is shown as a stacked bar graph.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Receptor-competition assay. Adult human articular chondrocytes were treated with or without indicated IL-6 family of cytokines and different doses of R805 for 4 hours. Co-IP measuring protein expression was performed after transfection with gp130 FLAG plasmid (72 hours) followed by western blot with respective receptor antibodies. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Competitive Binding Assay, Co-Immunoprecipitation Assay, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Receptor-competition assay. Adult human articular chondrocytes were treated with or without indicated IL-6 family of cytokines and different doses of R805 for 24 hours. Co-IP measuring protein expression was performed after transfection with gp130 FLAG plasmid (72 hours) followed by western blot with respective receptor antibodies. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Competitive Binding Assay, Co-Immunoprecipitation Assay, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Protein expression of gp130 pY814 in pig articular chondrocytes treated with or without OSM and R805 for 4 hours. Horizontal lines with bars show the mean ± SD. n=3. B. qPCR results from pig articular chondrocytes cells treated with OSM with or without R805 for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. C. Pig cartilage knee explants were incubated with or without OSM for 72 hours with or without R805 at indicated concentrations followed by a neoepitope assay. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Levels of complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of R805 for 24 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 nucleocapsid protein triggers hyperinflammation via protein-protein interaction-mediated intracellular Cl − accumulation in respiratory epithelium

doi: 10.1038/s41392-022-01048-1

Figure Lengend Snippet: SARS-CoV-2 N protein-elicited respiratory epithelial inflammation. a The mRNA expression levels of pro-inflammatory cytokines in N protein (50 μg/ml)-stimulated BEAS-2B cells ( n = 3). b Fluorescence labeling of β-tubulin (green) and N protein (red) in BEAS-2B cells with DAPI-labeled nuclei (blue) after stimulation with N protein (50 μg/ml) for 24 h. Scale bars, 10 μm. c The phosphorylation level of IκB and NF-κB p65 subunit after N protein (50 μg/ml) stimulation in BEAS-2B cells, with the gray value analysis ( n = 3). d Volcano plot visualization of the global RNA-seq DEGs in N protein (50 μg/ml)-stimulated BEAS-2B cells. e Dot plot of the enriched GO terms of the mRNAs exhibiting the enriched genes of BEAS-2B cells which were stimulated with N protein (50 μg/ml). f A heatmap showing the mRNA expression levels of the DEGs upregulated by N protein (50 μg/ml) stimulation compared with the control group, with the genes belonging to the GO annotations for cytokine activity and chemokine activity (GO: 0005125 and GO: 0008009). g The mRNA expression levels of cytokines in mice stimulated with N protein (0.25 mg/kg) ( n = 3). h The phosphorylation level of IκB and p65 after N protein (0.25 mg/kg) stimulation in mice, with the gray value analysis ( n = 3). i H&E staining of lung slices from N protein (0.25 mg/kg)-stimulated mice. Scale bars, 50 and 25 μm. j The mRNA expression levels of cytokines in SARS-CoV-2 (1 × 10 5 PFU)-infected hACE2-transduced mice ( n = 3). Data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control group or indicated by lines

Article Snippet: The samples were permeabilized with Triton x-100 for 30 min. After washing with cold PBS, the samples were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature, and incubated with the primary antibodies including anti-CFTR (sc-376683, 1:200, Santa Cruz Biotechnology), anti-phospho-SGK1 (36-002, 1:200, Millipore), anti-SARS-CoV-2 N protein (ab271180, rabbit mAb, 1:500, Abcam), anti-SARS-CoV-2 N protein (33717, mouse mAb, 1:400, CST), anti-Smad3 (9523, 1:100, CST), anti-pan-Keratin (4545, 1:500, CST), anti-Vimentin (EPR3776, 1:500, Abcam), anti-F4/80 (D4C8V) (30325, 1:500, CST), and anti-β IV Tubulin (bs-20694R, 1:200, Bioss, China) at 4 °C overnight.

Techniques: Expressing, Fluorescence, Labeling, RNA Sequencing Assay, Activity Assay, Staining, Infection