Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: a . The SPRBD- and SPRBD+IFNβ- induced increases in the mRNA expression of CHST15 and CHST11 were significantly reduced following exposure to SB20850, the p38-MAPK inhibitor. In contrast, NSC23766, a Rho/Rac-1 GTPase inhibitor, had no impact on their expression. b . SIS3, an inhibitor of phospho-Smad3, inhibited the SPRBD- and SPRBD+IFNβ- induced increases in expression of CHST15 and CHST11. c . Expression of ACE2 was inhibited by ACE2 specific siRNA following exposure to SPRBD alone or with IFNβ. d . Following silencing of ACE2 by siRNA, the SPRBD- and SPRBD+IFNβ- induced increases in phospho-T180/T182-p38 MAPK were inhibited. e . The SPRBD- and SPRBD+IFNβ- induced increases in phospho-S423/S425 SMAD3 were completely inhibited by SIS3 and, to a lesser extent, by SB20850, the p38 MAPK inhibitor. f . The SPRBD and SPRBD+IFNβ- induced increases in phospho-Thr180/Tyr182-p38 were unaffected by exposure to SIS3 and were inhibited by SB20850. g , h . Promoter activity of CHST15 and CHST11 was enhanced by exposure to SPRBD and to a greater extent by the combination of IFNβ and SPRBD. Both SIS3 and SB20850 inhibited promoter activation. [SIS3=specific inhibitor of SMAD3]. All p-values were determined by unpaired t-test, two-tailed, with unequal variance, and with at least three independent experiments. Error bars represent one standard deviation.

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Expressing, Activity Assay, Activation Assay, Two Tailed Test, Standard Deviation

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: a . ARSB mRNA expression was unaffected by NSC23766, but following exposure to the p38-MAPK inhibitor SB20850, mRNA expression was restored to baseline control values. b . In contrast, SIS3 had no impact on the SPRBD- or SPRBD+IFNβ-induced decline in ARSB expression. c . ARSB promoter activation was reduced by SPRBD and by SPRBD+IFNβ. These declines were reversed by exposure to SB20850, but not by SIS3. d . Following treatment with SPRBD or SPRBD+IFNβ, C-terminal phospho-(Ser807/811)-Rb declined as shown by ELISA. These declines were reversed by SB203580, but not by SIS3. e . In contrast to the decline in C-terminal Rb phosphorylation, exposure to SPRBD+IFNβ increased N-terminus phospho-(S249)-Rb, as detected by Western blot. This increase was inhibited by SB20850, and total Rb was unchanged. f . Densitometry confirms the impression of Western blot and shows the ratio of phospho-S249-Rb to total Rb following SPRBD+IFNβ has increased to 3.89 times the baseline. g . Following exposure to SPRBD and SPRBD+IFNβ, E2F-DNA binding declined significantly, and SB20850 reversed the declines. h . %DNA input declined following SPRBD+IFNβ and increased following inhibition of p38-MAPK by SB20850. i . Chromatin immunoprecipitation (ChIP) shows decline in E2F1 binding to the ARSB promoter following exposure to SPRBD+IFNβ; decline was reversed by SB203580. Agarose gel demonstrates no effect of the IgG negative control on E2F1 binding to the ARSB promoter at baseline, IFNβ control, reduced binding following SPRBD+IFNβ, and reversal of the decline following treatment with SB20850. j . Measurements of optical density confirm the impression from the gel. P-values were determined by unpaired t-tests, two-tailed with unequal variance, with n of at least 3 independent experiments. Error bars show one standard deviation.

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Inhibition, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Negative Control, Two Tailed Test, Standard Deviation

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: A . The antihistamine desloratadine reduced by 62% the SPRBD-induced increase in T180/T182 phospho-p38-MAPK in the AEC. B . Consistent with the decline in phospho-p38, desloratadine reduced the SPRBD-induced and SPRBD with IFNβ-induced increases in CHST15 (p=0.045, p=0.0041 with IFNβ) and CHST11 expression (p=0.008, p=0.0017 with IFNβ, unpaired t-tests, two-tailed, unequal variance, n=3). C, D . Consistent with the observed decline in ARSB, desloratadine partially reversed the SPRBD-induced decline in ARSB activity (from 64.7 to 73.4 nmol/mg protein/h and from 41.0 to 61.8 nmol/mg protein/h with IFNβ; n=3) and expression (0.71 to 0.87 and 0.46 to 0.74 with IFNβ, fold-change compared to control; n=3). E . The polyether antibiotic monensin, which modifies dermatan sulfate biosynthesis and processing [ - ], reduced the SPRBD-induced increases in CHST15 mRNA (p=0.003, p=0.006 with IFNβ) and CHST11 (p=0.003, p=0.002 with IFNβ; fold-change compared to control, unpaired t-tests, two-tailed, unequal variance). F . Monensin had no significant impact on the SPRBD-induced decline in ARSB.

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Expressing, Two Tailed Test, Activity Assay

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: Schematic of SARS-CoV-2 spike protein binding domain (SPRBD) peptide interaction and initiation of transcriptional events which modify chondroitin sulfation through activation of phospho-p38 MAPK. Both p38 MAPK and phsopho-Smad3 lead to increased expression of CHST15 and CHST11. AngII interaction with AT1 is also expected to increase phospho-p38. Phospho-p38 leads to N-terminal phosphorylation and activation of Rb with enhanced binding of E2F1, which reduces ARSB promoter activation due to reduced binding of E2F1. [ACE2=angiotensin converting enzyme 2; AngII=angiotensin II; ARSB=arylsulfatase B; AT1R=angiotensin II receptor type 1; AT2R=angiotensin II receptor type 2; CHST=carbohydrate sulfotransferase; pRb=retinoblastoma protein; SMAD=Suppressor of Mothers against Decapentaplegic; SPRBD=SARS-CoV-2 spike protein receptor binding domain]

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Protein Binding, Activation Assay, Expressing, Binding Assay

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: a . The SPRBD- and SPRBD+IFNβ- induced increases in the mRNA expression of CHST15 and CHST11 were significantly reduced following exposure to SB20850, the p38-MAPK inhibitor. In contrast, NSC23766, a Rho/Rac-1 GTPase inhibitor, had no impact on their expression. b . SIS3, an inhibitor of phospho-Smad3, inhibited the SPRBD- and SPRBD+IFNβ- induced increases in expression of CHST15 and CHST11. c . Expression of ACE2 was inhibited by ACE2 specific siRNA following exposure to SPRBD alone or with IFNβ. d . Following silencing of ACE2 by siRNA, the SPRBD- and SPRBD+IFNβ- induced increases in phospho-T180/T182-p38 MAPK were inhibited. e . The SPRBD- and SPRBD+IFNβ- induced increases in phospho-S423/S425 SMAD3 were completely inhibited by SIS3 and, to a lesser extent, by SB20850, the p38 MAPK inhibitor. f . The SPRBD and SPRBD+IFNβ- induced increases in phospho-Thr180/Tyr182-p38 were unaffected by exposure to SIS3 and were inhibited by SB20850. g , h . Promoter activity of CHST15 and CHST11 was enhanced by exposure to SPRBD and to a greater extent by the combination of IFNβ and SPRBD. Both SIS3 and SB20850 inhibited promoter activation. [SIS3=specific inhibitor of SMAD3]. All p-values were determined by unpaired t-test, two-tailed, with unequal variance, and with at least three independent experiments. Error bars represent one standard deviation.

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Expressing, Activity Assay, Activation Assay, Two Tailed Test, Standard Deviation

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: a . ARSB mRNA expression was unaffected by NSC23766, but following exposure to the p38-MAPK inhibitor SB20850, mRNA expression was restored to baseline control values. b . In contrast, SIS3 had no impact on the SPRBD- or SPRBD+IFNβ-induced decline in ARSB expression. c . ARSB promoter activation was reduced by SPRBD and by SPRBD+IFNβ. These declines were reversed by exposure to SB20850, but not by SIS3. d . Following treatment with SPRBD or SPRBD+IFNβ, C-terminal phospho-(Ser807/811)-Rb declined as shown by ELISA. These declines were reversed by SB203580, but not by SIS3. e . In contrast to the decline in C-terminal Rb phosphorylation, exposure to SPRBD+IFNβ increased N-terminus phospho-(S249)-Rb, as detected by Western blot. This increase was inhibited by SB20850, and total Rb was unchanged. f . Densitometry confirms the impression of Western blot and shows the ratio of phospho-S249-Rb to total Rb following SPRBD+IFNβ has increased to 3.89 times the baseline. g . Following exposure to SPRBD and SPRBD+IFNβ, E2F-DNA binding declined significantly, and SB20850 reversed the declines. h . %DNA input declined following SPRBD+IFNβ and increased following inhibition of p38-MAPK by SB20850. i . Chromatin immunoprecipitation (ChIP) shows decline in E2F1 binding to the ARSB promoter following exposure to SPRBD+IFNβ; decline was reversed by SB203580. Agarose gel demonstrates no effect of the IgG negative control on E2F1 binding to the ARSB promoter at baseline, IFNβ control, reduced binding following SPRBD+IFNβ, and reversal of the decline following treatment with SB20850. j . Measurements of optical density confirm the impression from the gel. P-values were determined by unpaired t-tests, two-tailed with unequal variance, with n of at least 3 independent experiments. Error bars show one standard deviation.

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Inhibition, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Negative Control, Two Tailed Test, Standard Deviation

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: A . The antihistamine desloratadine reduced by 62% the SPRBD-induced increase in T180/T182 phospho-p38-MAPK in the AEC. B . Consistent with the decline in phospho-p38, desloratadine reduced the SPRBD-induced and SPRBD with IFNβ-induced increases in CHST15 (p=0.045, p=0.0041 with IFNβ) and CHST11 expression (p=0.008, p=0.0017 with IFNβ, unpaired t-tests, two-tailed, unequal variance, n=3). C, D . Consistent with the observed decline in ARSB, desloratadine partially reversed the SPRBD-induced decline in ARSB activity (from 64.7 to 73.4 nmol/mg protein/h and from 41.0 to 61.8 nmol/mg protein/h with IFNβ; n=3) and expression (0.71 to 0.87 and 0.46 to 0.74 with IFNβ, fold-change compared to control; n=3). E . The polyether antibiotic monensin, which modifies dermatan sulfate biosynthesis and processing [ - ], reduced the SPRBD-induced increases in CHST15 mRNA (p=0.003, p=0.006 with IFNβ) and CHST11 (p=0.003, p=0.002 with IFNβ; fold-change compared to control, unpaired t-tests, two-tailed, unequal variance). F . Monensin had no significant impact on the SPRBD-induced decline in ARSB.

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Expressing, Two Tailed Test, Activity Assay

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: Schematic of SARS-CoV-2 spike protein binding domain (SPRBD) peptide interaction and initiation of transcriptional events which modify chondroitin sulfation through activation of phospho-p38 MAPK. Both p38 MAPK and phsopho-Smad3 lead to increased expression of CHST15 and CHST11. AngII interaction with AT1 is also expected to increase phospho-p38. Phospho-p38 leads to N-terminal phosphorylation and activation of Rb with enhanced binding of E2F1, which reduces ARSB promoter activation due to reduced binding of E2F1. [ACE2=angiotensin converting enzyme 2; AngII=angiotensin II; ARSB=arylsulfatase B; AT1R=angiotensin II receptor type 1; AT2R=angiotensin II receptor type 2; CHST=carbohydrate sulfotransferase; pRb=retinoblastoma protein; SMAD=Suppressor of Mothers against Decapentaplegic; SPRBD=SARS-CoV-2 spike protein receptor binding domain]

Article Snippet: Phospho-p38 MAPK was measured in cell samples using a Pathscan® Phospho-p38 MAPK Thr180/Tyr182 Sandwich ELISA (Cell Signaling).

Techniques: Protein Binding, Activation Assay, Expressing, Binding Assay

Journal: Physiological Reports

Article Title: Interleukin‐38 suppresses abdominal aortic aneurysm formation in mice by regulating macrophages in an IL1RL2 ‐p38 pathway‐dependent manner

doi: 10.14814/phy2.15581

Figure Lengend Snippet: The mechanism how interleukin‐38 suppresses macrophage activation. (a) Representative western blot analysis of p‐p38, p38, p‐JNK, and JNK in the aortic wall on day 14. (b) Bar graphs showing the relative densitometry of p‐p38 ( n = 6, p = 0.0280 [ t test], p = 0.0411 [Mann–Whitney U test]) and p‐JNK ( n = 6, p = 0.6893 [ t test], p = 0.6991 [Mann–Whitney U test]). (c) Representative western blot analysis of p‐p38, p38, p‐JNK, and JNK in macrophages after TNF‐α stimulation with or without IL‐38. (d) Bar graphs showing the relative densitometry of p‐p38 ( n = 6, p < 0.0001 [ANOVA], p < 0.0001 [post hoc, Control vs. TNF‐α], p = 0.0234 [post hoc, Control vs. TNF‐α + IL‐38], p = 0.0125 [post hoc, TNF‐α vs. TNF‐α + IL‐38]) and p‐JNK ( n = 6, p = 0.0283 [ANOVA], p = 0.0264 [post hoc, Control vs. TNF‐α]). (e) Representative western blot analysis of iNOS, MMP‐9, MMP‐2, and GAPDH in macrophages after TNF‐α stimulation with or without IL‐38 in the presence of SB203580. (f) Bar graphs showing the relative densitometry of iNOS ( n = 3, p = 0.2000 [Mann–Whitney U test]), MMP‐9 ( n = 3, p = 0.7000 [Mann–Whitney U test]), and MMP‐2 ( n = 3, p = 0.7000 [Mann–Whitney U test]). (g) Representative photomicrographs of the aortic wall on day 14 in the presence of SB203580, stained with EVG (upper row) or anti‐CD68 antibody (lower row). (h) Bar graph showing the number of CD68 + cells in the aortic wall on day 14 in the presence of SB203580 ( n = 5–6, p = 0.6623 [Mann–Whitney U test]). (i) Bar graphs showing the aorta diameter ( n = 5–6, p = 0.2468 [Mann–Whitney U test]), AAA incidence ( n = 5–6, p = 0.2506 [χ 2 test]), adventitia ( n = 5–6, p > 0.9999 [Mann–Whitney U test]), intramedia ( n = 5–6, p = 0.1710 [Mann–Whitney U test]), and elastin degradation score ( n = 5–6, p = 0.4917 [ t test], p = 0.6753 [Mann–Whitney U test]) on day 14 in the presence of SB203580.

Article Snippet: Primary antibodies included anti‐TNF‐α (rabbit monoclonal, dilution 1:400, #11948; Cell Signaling Technology), anti‐iNOS (rabbit monoclonal, dilution 1:400, #13120; Cell Signaling Technology), anti‐ MMP‐9 (rabbit polyclonal, dilution 1:1000, #ab38898; abcam), anti‐ MMP‐2 (rabbit polyclonal, dilution 1:1000, #ab97779; abcam), anti‐IL1RL2 (rabbit polyclonal, dilution 1:1000, # PA5‐38013; Invitrogen), anti‐p38 (rabbit polyclonal, dilution 1:5000, #9212; Cell signaling technology), anti‐phospho‐p38 (p‐p38) (rabbit polyclonal, dilution 1:1000, #9211; Cell signaling technology), anti‐JNK (rabbit polyclonal, dilution 1:5000, #9252; Cell signaling technology) and anti‐phospho‐JNK (p‐JNK) (rabbit polyclonal, dilution 1:1000, #9251; Cell signaling technology).

Techniques: Activation Assay, Western Blot, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: EVIDENCE FOR ANGIOTENSIN II AS A NATURALLY EXISTING SUPPRESSOR FOR THE NATRIURETIC PEPTIDE SYSTEM

doi: 10.1101/2023.01.26.525806

Figure Lengend Snippet: (A) Protein expression of different human PKC isoforms in the membrane and cytosol fractions of different HEK293 transfected cell lines (with or without treatment of ANGII). GC-A and NaK-ATPase serve as quality control for the separation of membrane and cytosol fractions. GAPDH serves as loading control. (B) Protein expression of phosphorylated PKC substrates, phosphorylated p38 MAPK, and p38 MAPK in HEK293/GC-A + /AT 1 + cells treated with different PKC modulators. GAPDH serves as loading control. PMA, phorbol 12-myristate 13-acetate. (C) In vitro cGMP generation in HEK293/GC-A + /AT 1 + in response to ANP (10 −8 M) with or without ANGII (10 −8 M) and valsartan (1 μM) or Go6983 (5 μM). Values of cGMP were normalized to vehicle group (blue) under ANP treatment. * indicates P <0.05, one-way ANOVA with Dunnett multiple comparisons test. (D) Hypothetical mechanism underlying the crosstalk between ANGII and GC-A derived from our in vitro studies.

Article Snippet: The following primary antibodies were used: anti-GCA (catalog#MAB48601, R&D System, Minneapolis, MN) at 1:1000 dilution, anti-GCB (catalog#55113-1-AP, Proteintech, Rosemont, IL) at 1:200 dilution, anti-GFP (catalog#TA150041, OriGene, Rockville, MD) at 1:1000 dilution, anti-PKCα (catalog#2056, Cell Signaling Technology, Danvers, MA) at 1:500 dilution, anti-PKCε (catalog#2683, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, anti-Phspho-(Ser) PKC Substrate (catalog#2261, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, anti-NaK-ATPase (catalog#EP1845Y, abcam, Waltham, MA) at 1:2000 dilution, anti-p38 MAPK (catalog#8690, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, anti-Phospho-p38 MAPK (catalog#4511, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, and anti-GAPDH (catalog#2118, Cell Signaling Technology, Danvers, MA) at 1:5000 dilution.

Techniques: Expressing, Transfection, In Vitro, Derivative Assay

Journal: PLOS Neglected Tropical Diseases

Article Title: Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways

doi: 10.1371/journal.pntd.0011062

Figure Lengend Snippet: Details of the antibodies used in this study.

Article Snippet: Phospho-p38 , Rabbit monoclonal , Rabbit IgG , 1:1000 , Cell Signaling Technology.

Techniques:

Journal: PLOS Neglected Tropical Diseases

Article Title: Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways

doi: 10.1371/journal.pntd.0011062

Figure Lengend Snippet: (A, B) The phosphorylation of AKT, p65, ERK and p38 in liver were analyzed by Western blot. (C, D) 3 × 10 5 WT and TLR2 -/- mouse BECs were cultivated with C . sinensis ESPs (50 μg/mL) for 120 min, the cells were lysed in cells lysates and the phosphorylation of AKT, p65, ERK and p38 were analyzed by Western blot. (E) The production levels of IL-6 in the supernatant of BECs pretreated with or without AKT inhibitor, p38 inhibitor and ERK inhibitor for 1 h, then co-cultured with ESPs for 18 h were measured by ELISA.

Article Snippet: Phospho-p38 , Rabbit monoclonal , Rabbit IgG , 1:1000 , Cell Signaling Technology.

Techniques: Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLOS Neglected Tropical Diseases

Article Title: Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways

doi: 10.1371/journal.pntd.0011062

Figure Lengend Snippet: C . sinensis triggered AKT and p38 signal pathways dependent on TLR2 of BECs to promote the production of IL-6, which subsequently activated TGF-β1-Smad2/Smad3 pathways, myofibroblasts and up-regulated the expression of ECMs, finally resulting in biliary fibrosis in mice. In the figure, ‘ ↑ ’indicates positive regulation.

Article Snippet: Phospho-p38 , Rabbit monoclonal , Rabbit IgG , 1:1000 , Cell Signaling Technology.

Techniques: Expressing