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: Experimental gerontology

Article Title: Aerobic exercise modulates aortic chondrogenesis and calcification via 5-methoxytryptophan and P38MAPK in atherosclerotic rats.

doi: 10.1016/j.exger.2025.112722

Figure Lengend Snippet: Fig. 7. B–F indicates the effects of aerobic exercise on aortic P38MAPK, pp38MAPK, pp38MAPK/P38MAPK, collagen II, and IL-6 protein expression in AS rats, compared with Con group, * p < 0.05 and **p < 0.01; compared with AS group # p < 0.05 and ##p < 0.01.

Article Snippet: The PVDF transmembrane was transfected with the addition of primary antibodies pp38 MAPK (1:1000, ab4822, abcam), p38 MAPK (1:1000, NB100–56446, NOVUS), collagen II (1:1000, ab34712, abcam), IL-6 (1:1000, NBP2–89149, NOVUS), incubated for 12 h, secondary antibody incubated for 2 h, ECL developed, fixed, exposed and scanned to process the gray value of each band.

Techniques: Expressing

Journal: Molecular psychiatry

Article Title: n-3 polyunsaturated fatty acid deprivation in rats decreases frontal cortex BDNF via a p38 MAPK-dependent mechanism.

doi: 10.1038/sj.mp.4001888

Figure Lengend Snippet: Figure 3 Frontal cortex PKA (a), CaMK IV (b), PKC (c) and p44 MAPK (d) activities in n-3 PUFA þ and n-3 PUFA rats. Representative frontal cortex nuclear immunoblots of phosphorylated MAPK p42 and p44 protein levels in n-3 PUFA þ and n-3 PUFA rats (e). Data were compared using unpaired t-tests, mean7s.e.m., n = 10 independent samples per group, *P < 0.05.

Article Snippet: Nuclear extracts protein blots were incubated overnight with antiphosphorylated p42 or p44 MAPK (Santa Cruz) (1:200 dilution) or antiphosphorylated p38 MAPK antibody (R&D Systems, 1:200 dilution), followed by HRPconjugated secondary antibodies (Bio-Rad).

Techniques: Western Blot

Journal: Molecular psychiatry

Article Title: n-3 polyunsaturated fatty acid deprivation in rats decreases frontal cortex BDNF via a p38 MAPK-dependent mechanism.

doi: 10.1038/sj.mp.4001888

Figure Lengend Snippet: Figure 4 Frontal cortex cytosolic p38 MAPK activity in n-3 PUFA þ and n-3 PUFA rats (a). Phosphorylated p38 MAPK protein levels in n-3 PUFA þ and n-3 PUFA rats (b). Data were compared using unpaired t-tests, mean7 s.e.m., n = 10 independent samples per group, *P < 0.05.

Article Snippet: Nuclear extracts protein blots were incubated overnight with antiphosphorylated p42 or p44 MAPK (Santa Cruz) (1:200 dilution) or antiphosphorylated p38 MAPK antibody (R&D Systems, 1:200 dilution), followed by HRPconjugated secondary antibodies (Bio-Rad).

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Computational and In Vitro Analysis of Plumbagin’s Molecular Mechanism for the Treatment of Hepatocellular Carcinoma

doi: 10.3389/fphar.2021.594833

Figure Lengend Snippet: (A) Protein expression levels of LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK in SMMC-7721 cells measured by Western blot after treatment with 5 μM PL, 10 μM 3-MA autophagy inhibitor, 10 μM rapamycin autophagy agonist, 10 μM SB202190, and 10 μM SB203580 p38 MAPK inhibitor. (B) LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK were used as internal reference total proteins for gray value analysis. * p < 0.05, ** p < 0.01 *** p < 0.001, compared with the PL group. # p < 0.05, ## p < 0.01 ### p < 0.001 vs. the control group. (C) Protein expression levels of LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK in BEL-7404 cells measured by Western blot after treatment with 5 μM PL, 10 μM 3-MA autophagy inhibitor, 10 μM rapamycin autophagy agonist, 10 μM SB202190, and 10 μM SB203580 p38 MAPK inhibitor. (D) LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK were used as internal reference total proteins for gray value analysis. * p < 0.05, ** p < 0.01 *** p < 0.001, compared with the PL group. # p < 0.05, ## p < 0.01 ### p < 0.001 vs. the control group.

Article Snippet: Plumbagin (PL) was purchased from Sigma-Aldrich (St. Louis, MO, United States) with purity ≥98%; the Acridine Orange (AO)/Ethidium bromide (EB) Double Stain Kit was from Solable Technology (Beijing, China); N-acetyl-l-cysteine, SB203580, and SB202190 were from Sigma-Aldrich (St. Louis, MO, United States); SC-79, MEK2206, 3-MA, and Z-VAD-FMK were from Selleck (Texas, United States); the BCA Protein Assay Kit, ROS Assay Kit, Annexin V-FITC Apoptosis Detection Kit and Cell lysis buffer for Western were all obtained from Beyotime Biotechnology (Shanghai, China); antibodies against Akt, phospho-Akt, mTOR, phospho-mTOR, p38 MAPK, phospho-p38 MAPK, PI3K, phospho-PI3K, LC3B, cleave-RP, and cleave-caspase 3 were from Cell Signaling Technology, Inc. (Boston, MA, United States); and β-p38 MAPK was purchased from Boster Technology (Wuhan, China).

Techniques: Expressing, Western Blot, Control