Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel ( B ) shows that in Agtr1a -/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na + /HCO 3 - cotransporter expression in wild-type mPCT cells. Panel ( A ) shows that ECFP/Ang II significantly increased Na + /HCO 3 - expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT 1 receptors in mPCT cells. Panel ( B ) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na + /HCO 3 - expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT 1 and AT 2 receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel ( B ) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a -/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel ( C ) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a -/- mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a -/- mPCT cells transfected with ECFP/ANG II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: SPARC activated p38γ signaling to stabilize PFKFB3 protein expression. A , B GSEA manifested that SPARC regulated p38 pathway. C The interaction between SPARC, p38γ, and PFKFB3 in PPI network. D After transfected with pcDNA-SPARC, si-SPARC-1#, and si-SPARC-2#, p38γ and PFKFB3 expression was measured via western blot. E After transfected with si-p38γ or treated with SB203580, PFKFB3 expression was measured via western blot. F Western blot of co-IP was performed to verify the binding ability between p38γ and PFKFB3 in KFs. G After transfected with si-p38γ and treated with CHX, p38γ and PFKFB3 expression in KFs was measured via western blot. H Immunohistochemical staining and I western blotting for p38γ and PFKFB3 in human keloids and extra-lesional samples. ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Immunohistochemical staining, Staining

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: SPARC promotes the proliferation, migration, collagen production, and glycolysis of KFs via regulating p38γ signaling. After transfected with pcDNA-SPARC and si-p38γ, and treated with SB203580, ECAR ( A ), OCR ( B ), glucose uptake ( C ), and lactate production ( D ) were investigated in KFs. PFKFB3 expression was measured via western blot ( E ). The proliferation and migration were assessed using EdU ( F ) and transwell assay ( G ). α-SMA, Fibronectin, Collagen I, and Collagen III expression was measured via western blot ( H ). * P < 0.05, ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Migration, Transfection, Expressing, Western Blot, Transwell Assay

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: SPARC, p38γ, and PFKFB3 were increased in the skin of BLM-induced fibrosis mouse model. A Immunohistochemical staining for SPARC, p38γ, and PFKFB3. B Western blot for assessing SPARC, p38γ, and PFKFB3 expression. ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Immunohistochemical staining, Staining, Western Blot, Expressing

Journal: Inflammation and Regeneration

Article Title: SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

doi: 10.1186/s41232-024-00357-y

Figure Lengend Snippet: Inhibition of p38γ and PFKFB3 relieved BLM-induced skin fibrosis in vivo. After injected with SB203580 and 3PO, p38γ and PFKFB3 expression in BLM-induced mice skin tissues was measured employing western blot ( A ). The dermal thickness and collagen thickness of mice skin tissues were detected utilizing H&E and Masson’s trichrome staining ( B ). Immunohistochemical staining for α-SMA and Ki67 in BLM-induced mice skin tissues ( C ). * P < 0.05, ** P < 0.01

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the primary antibodies in 5% milk-TBST overnight at 4 °C using the following concentrations: SPARC (1:500, no. ab290636 and ab225716, Abcam, UK), p38γ (1:1000, no. 20184–1-AP, Proteintech, USA), PFKFB3 (1:1000, no. 13763–1-AP, Proteintech, USA), α-SMA (1:1000, no. #19245, Cell Signaling Technology, USA), Fibronectin (1:500, no. ab2413, Abcam, UK), Collagen I (1:500, no. ab138492, Abcam, UK), Collagen III (1:500, no. ab184993, Abcam, UK) and β-actin (1:2000, no. ab8227, Abcam, UK).

Techniques: Inhibition, In Vivo, Injection, Expressing, Western Blot, Staining, Immunohistochemical staining

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: IL-33 decreases I A via p38 MAPK. A, effects of 50 ng/mL IL-33 on phospho-Akt ( p -Akt) or total Akt ( t -Akt) protein abundance in the presence or absence of Akt inhibitor III (Akt-III, 10 µM) in DRG cells. Blots are representative of three independent experiments with β-tubulin serving as a loading control. ** p < 0.01 vs. control, unpaired t test. B, colabeling (white arrows) of ST2 and Akt and p38 in mouse DRG sections. Scale bar, 50 µm. C-D, time course of I A changes indicating the effects of IL-33 on I A in the presence of 10 µM Akt inhibitor III ( C ) or 20 µM LY294002 ( D ). Arabic numerals indicate the points utilized for the example current traces. E, bar graph showing the effects of 50 ng/mL IL-33 on I A in the presence of Akt inhibitor III ( n = 10 cells) or LY294002 ( n = 8 cells) as indicated in Panels B and C , respectively. Application of 10 µM Akt inhibitor III ( n = 6 cells) or 20 µM LY294002 ( n = 6 cells) alone did not significantly affect I A . F, effects of 50 ng/mL IL-33 on p -p38, p -JNK and p -ERK protein abundance in DRG cells. Blots are representative of three independent experiments with β-tubulin serving as a loading control. * p < 0.05 vs. control, unpaired t test. G, pretreatment of DRG cells with the ST2 neutralizing antibody (ST2 Ab, 2 µg/mL) or R406 (1 µM) abolished the 50 ng/mL IL-33-induced increase in p -p38 protein abundance. Blots are representative of three independent experiments with β-tubulin serving as a loading control. H, time course of I A changes indicating the effect of 50 ng/mL on I A in cells pretreated with 10 µM SB203580. I, bar graph showing that pretreating cells with SB203580 ( n = 10 cells), but not its inactive analogue SB202474 (10 µM, n = 6 cells), prevented the IL-33-induced I A decrease. ** p < 0.01 vs. control, paired t test.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Quantitative Proteomics, Control

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: p38β mediates the IL-33-induced I A decrease. A, immunoblot analysis of p38α and p38β protein abundance in DRGs. Mouse brains were used as positive controls. Blots are representative of three independent experiments with β-tubulin serving as a loading control. B, time course of I A changes ( left ) and bar graph ( right ) indicating the effect of 50 ng/mL IL-33 on I A in the presence of JX-401 (50 nM, n = 8 cells). The application of 50 nM JX-401 ( n = 6 cells) alone had no significant effect on I A . Arabic numerals indicate the points utilized for the example current traces. C, immunoblot analysis showing that the protein expression level of p38β was significantly reduced in the p38β-siRNA-treated groups, while the expression of p38α was not affected. Blots are representative of three independent experiments with β-tubulin serving as a loading control. ** p < 0.01 vs. NC-siRNA, unpaired t test. D, example traces ( left ) and bar graph ( right ) demonstrating the effects of 50 ng/mL IL-33 on I A in cells treated with control siRNA (NC-siRNA, n = 9 cells) or p38β-siRNA ( n = 11 cells). ** p < 0.01 vs. control + NC-siRNA group, one-way ANOVA with a Bonferroni post hoc test.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Western Blot, Quantitative Proteomics, Control, Expressing

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: IL-33/ST2 signaling participates in peripheral pain sensitivity. A-B, intraplantar injection (i.p.l.) of IL-33 at 30 ng, 100 ng, and 300 ng significantly decreased the mechanical paw withdrawal threshold (PWT, A ) and heat paw withdrawal latency (PWL, B ). * p < 0.05, ** p < 0.01, *** p < 0.001, vs. vehicle at the corresponding points, two-way ANOVA with a Bonferroni post hoc test. C-D, intraplantar pretreatment with 1 µg of ST2 neutralizing antibody (ST2 Ab) completely prevented the mechanical ( C ) and heat ( D ) hypersensitivity induced by 100 ng of IL-33 (i.p.l.). *** p < 0.001 vs. vehicle at 3 h, two-way ANOVA with a Bonferroni post hoc test. E-F, p38β siRNA attenuated mechanical and heat hypersensitivity induced by 100 ng of IL-33 (i.p.l.). *** p < 0.001 vs. vehicle at 3 h, # P < 0.05, ## P < 0.01 vs. vehicle in the NC-siRNA groups, two-way ANOVA with a Bonferroni post hoc test. G-H, intraplantar pretreatment with 25 nmol 4-AP occluded mechanical ( G ) and heat ( H ) hypersensitivity mediated by 100 ng of IL-33. *** p < 0.001 vs. vehicle at 3 h, two-way ANOVA with a Bonferroni post hoc test. I-J, intraplantar injection of sST2 at 2 µg attenuated the mechanical hypersensitivity ( I ) and thermal hyperalgesia ( J ) in CFA mice. The arrow indicates the injection of sST2 or vehicle. *** p < 0.001 vs. vehicle at the corresponding points, two-way ANOVA with a Bonferroni post hoc test. K-L, representative current traces ( K ) and summary data ( L ) indicating that intraplantar injection of sST2 (2 µg) abolished the CFA (2 d)-induced I A decrease in small-sized DRG neurons ( n = 11-14 neurons per group). ** p < 0.01 compared with the normal saline (NS) group, ## p < 0.01 compared with the CFA + vehicle group, one-way ANOVA with a Bonferroni post hoc test. N = at least 7 mice for all animal behavior experiments.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Injection, Saline

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: Schematic showing the molecular mechanism of IL-33-induced neuronal hyperexcitability of DRG neurons and pain hypersensitivity in mice. IL-33 acting through ST2 receptors does not affect the activity of JAK2 but leads to the activation of Syk. The increased level of p -Syk stimulates downstream p38β signaling, which in turn regulates A-type channel activity and results in I A reduction. IL-33/ST2-mediated signaling enhances neuronal excitability of DRG neurons and nociceptive behaviors in mice. Neither PKA nor PI3K/Akt was necessary for the IL-33-induced I A response in this study. Whether p38β directly phosphorylates the channels encoding I A or stimulates intermediate molecules in small DRG neurons needs to be investigated further. Created with BioRender.com.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Activity Assay, Activation Assay

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Scientific Reports

Article Title: Cellular response to persistent foot-and-mouth disease virus infection is linked to specific types of alterations in the host cell transcriptome

doi: 10.1038/s41598-018-23478-0

Figure Lengend Snippet: Differential expression of MAPK signaling-related genes in BHK-VECs. ( a ) qRT-PCR validation of DEGs related to MAPK pathways in BHK-VECs. GAPDH served as the internal reference gene to normalize data, and three biologically independent replicates were performed. ( b , c ) Effect of MAPK/ERK or p38/MAPK inhibition on replication of FMDV in BHK-21 cells. BHK-21 cells were pre-incubated (1 h) with DMSO, ( b ) 20 or 50 mM U0126, or ( c ) 20 or 50 mM SB202190 and then infected with FMDV at 2.5 × 10 −4 PFU/cell for 24 h in the presence of DMSO, U0126, or SB202190. Protein extracts were examined using western blotting with FMDV 3D-specific or the indicated antibodies; the effectiveness of inhibition was monitored by detecting the phosphorylation of inhibitor-specific target protein (P-MAPK/ERK and pS15-Hsp27). ( d ) (i) p38δ (MAPK13) was down-regulated in BHK-VECs compared with BHK-21 cells, and (ii) overexpression of MAPK13 genes in BHK-21 cells promoted the replication of FMDV.

Article Snippet: GAPDH antibody, MAPK13 antibody, Dcn antibody, Ccnd1 antibody, and secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit and goat anti-mouse antibodies were purchased from Proteintech.

Techniques: Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery, Inhibition, Incubation, Infection, Western Blot, Phospho-proteomics, Over Expression

Journal: BMC Endocrine Disorders

Article Title: The role of MAPK11/12/13/14 (p38 MAPK) protein in dopamine agonist-resistant prolactinomas

doi: 10.1186/s12902-021-00900-9

Figure Lengend Snippet: Bromocriptine upregulates DRD2 levels and reduces MAPK11/12/13/14 and PRL levels in rat prolactinomas. a Analysis of the appearance of the pituitary in the Control, Prolactinoma, and BRC groups. b Ratio of pituitary weight to body weight in rats, **** P < 0.0001 vs. Control; #### P < 0.0001 vs. Prolactinoma, ( n = 10). c Detection of serum PRL level in rats by ELISA. *** P < 0.001 vs. Control; ### P < 0.001 vs. Prolactinoma,( n = 6). d The protein expression levels of DRD2, MAPK11, MAPK12, MAPK13, MAPK14 and PRL in rat pituitary were detected by western blot. e The expression levels of DRD2, MAPK11, MAPK12, MAPK13, MAPK14 and PRL mRNA in rat pituitary were detected by RT-qPCR. * p < 0.05 vs. control, *** p < 0.001 vs. control, # p < 0.05 vs. prolactinoma, ### p < 0.001 vs. Prolactinoma,( n = 3)

Article Snippet: Oestradiol benzoate injection was purchased from Sichuan Jinke Pharmaceutical Co., Ltd. Anti-dopamine D2 receptor (D2R; cat. no. 55084–1-AP), MAPK11 (cat. no. 17376–1-AP), MAPK12 (cat. no. 20184–1-AP), MAPK13 (cat. no. 10217–1-AP), MAPK14 (cat. no. 14064–1-AP), Bcl2 (cat. no. 26593–1-AP) and Bax (cat. no. 50599–2-lg) primary antibodies were purchased from Proteintech. β-actin (cat. no. T0022), PRL (cat. no. DF6506) and NF-κB p65 (cat. no. BF0382) antibodies were purchased from Affinity.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Quantitative RT-PCR

Journal: BMC Endocrine Disorders

Article Title: The role of MAPK11/12/13/14 (p38 MAPK) protein in dopamine agonist-resistant prolactinomas

doi: 10.1186/s12902-021-00900-9

Figure Lengend Snippet: Comparison of the effects of bromocriptine on MAPK11/12/13/14 and PRL in GH3 cells and MMQ cells. a , b Western blot analysis of the protein expression of MAPK11, MAPK12, MAPK13, MAPK14 and PRL after treating GH3 cells and MMQ cells with different concentrations of bromocriptine(12.5 μM, 25 μM, 50 μM, 100 μM) for 48 h. * p < 0.05 vs. 0 μM, ** p < 0.01 vs. 0 μM, *** p < 0.001 vs. 0 μM, **** p < 0.0001 vs. 0 μM, ( n = 3). c RT-qPCR analysis of MAPK11, MAPK14 and PRL mRNA expression after bromocriptine treatment of GH3 cells and MMQ cells for 48 h. ** p < 0.01 vs. 0 μM, *** p < 0.001 vs. 0 μM, **** p < 0.0001 vs. 0 μM, ( n = 3)

Article Snippet: Oestradiol benzoate injection was purchased from Sichuan Jinke Pharmaceutical Co., Ltd. Anti-dopamine D2 receptor (D2R; cat. no. 55084–1-AP), MAPK11 (cat. no. 17376–1-AP), MAPK12 (cat. no. 20184–1-AP), MAPK13 (cat. no. 10217–1-AP), MAPK14 (cat. no. 14064–1-AP), Bcl2 (cat. no. 26593–1-AP) and Bax (cat. no. 50599–2-lg) primary antibodies were purchased from Proteintech. β-actin (cat. no. T0022), PRL (cat. no. DF6506) and NF-κB p65 (cat. no. BF0382) antibodies were purchased from Affinity.

Techniques: Comparison, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 2. Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 M). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 3. Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-B activation. A, HEK293 cells were left untreated, or were trans- fected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simul- taneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si- Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8- reporter (B) or NF-B reporter construct (C and D) and a -galactosi- dase reporter plasmid. Cells were either left untreated () or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were tran- siently transfected with a control vector (ctrl) or NOD2 expression plas- mid along with an IL-8 luciferase reporter plasmid (E) or a NF-B- driven luciferase reporter (F), respectively, and a -galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild- type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively ac- tive Rac1L61. Cells were either stimulated with 10 g/ml MDP (MDP) or left untreated (), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-B-driven luciferase reporter and a -galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or con- stitutive active Rac1 (Rac1L61) mean SD; , p 0.01.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Activation Assay, Control, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Luciferase, Dominant Negative Mutation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 6. Involvement of -PIX in NOD2-medi- ated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of -Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting -Pix (si--Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti--Pix Abs were performed. Western blots were si- multaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 g/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti- NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data pre- sented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Sequencing, Western Blot, Incubation, Membrane, Enzyme-linked Immunosorbent Assay