Journal: Nature Communications

Article Title: Cisplatin toxicity is counteracted by the activation of the p38/ATF-7 signaling pathway in post-mitotic C. elegans

doi: 10.1038/s41467-023-38568-5

Figure Lengend Snippet: a Western blot analysis after SDS-PAGE of phospho-p38 levels in 1-day-old wild-type adults with and without cisplatin treatment for indicated times. Worms were exposed to cisplatin on 300 μg/mL cisplatin-containing plates. The blot was probed with anti:phospho-p38 antibody to detect activated PMK-1, anti:PMK-1 antibody was used to estimate total levels of PMK-1 protein, tubulin was used as a loading control. Full uncropped images are available in Supplementary Fig. . b Relative phospho-p38 level quantification from blots presented in Supplementary Fig. . Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. Bars represent mean ± SD. c Western blot analysis after SDS-PAGE of phospho-p38 levels in 1-day-old wild-type adults with (+) and without (−) MitoTempo/cisplatin exposure. The blot was probed with anti:phospho-p38 antibody, tubulin was used as a loading control. Full uncropped images are available in Supplementary Fig. . d Relative phospho-p38 levels quantification from blots presented in Supplementary Fig. . Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. Bars represent mean ± SD. e , h , i Mean survival ± SD of 1-day-old adults with the indicated genotypes after 24 h cisplatin (300 μg/mL) exposure. Statistical significance determined was by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. All data on cisplatin sensitivity trials is available in Supplementary Data . f , g Relative gene expression of four phase II detoxification genes in 1-day-old adult wild-type animals ( f ) without or with (+CP) 6 h or 24 h cisplatin treatment, g without or with (+CP) 6 h cisplatin treatment. Worms were exposed to cisplatin on 300 μg/mL cisplatin-containing plates. Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate for 6 h exposure timepoint and duplicate for 24 h exposure timepoint. F44B9.5 was used as a normalizing control. Bars represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were: anti:phospho-p38 MAPK (Thr180/Thr182) Rabbit mAb (D3F9, Cell Signaling) 1:1000, anti:total PMK-1 MAPK Rabbit pAb 1:1000 from the Pukkila-Worley lab , anti:tubulin (T5168, Sigma) 1:5000, anti:Flag (M2, Sigma) 1:1000.

Techniques: Western Blot, SDS Page, Expressing

Journal: Nature Communications

Article Title: Cisplatin toxicity is counteracted by the activation of the p38/ATF-7 signaling pathway in post-mitotic C. elegans

doi: 10.1038/s41467-023-38568-5

Figure Lengend Snippet: a Western blot analysis after SDS-PAGE of phospho-p38 levels in 1-day-old adults with (+) and without (–) 1 mM auxin (AUX) treatment. The blot was probed with anti:phospho-p38 antibody and tubulin was used as a loading control. Full uncropped images are available in Supplementary Fig. . b , d Mean survival ± SD of 1-day-old adults with the indicated genotypes after 24 h exposure to 300 μg/mL cisplatin-containing plates without (−AUX) or with (+AUX) 1 mM auxin treatment (see the “Methods” section for details). Statistical significance was determined by the b independent two-sided t -test or d one-way ANOVA followed by Bonferroni post hoc correction. The experiment was performed in triplicate. c Western blot analysis after SDS-PAGE of SEK-1 levels in 1-day-old adults with (+) and without (−) 1 mM auxin (AUX) treatment. The blot was probed with anti:Flag antibody and tubulin was used as a loading control. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were: anti:phospho-p38 MAPK (Thr180/Thr182) Rabbit mAb (D3F9, Cell Signaling) 1:1000, anti:total PMK-1 MAPK Rabbit pAb 1:1000 from the Pukkila-Worley lab , anti:tubulin (T5168, Sigma) 1:5000, anti:Flag (M2, Sigma) 1:1000.

Techniques: Western Blot, SDS Page

Journal: Frontiers in Immunology

Article Title: Streptococcus Suis Serotype 2 Stimulates Neutrophil Extracellular Traps Formation via Activation of p38 MAPK and ERK1/2

doi: 10.3389/fimmu.2018.02854

Figure Lengend Snippet: Role of the activation of p38 MAPK and ERK1/2 in SS2 and PMA induced NETs formation. (A) Neutrophils were infected with SS2 ZY05719 or treated with PMA for 2 h; p38 MAPK and ERK1/2 phosphorylation were determined with western blotting using specific antibody against phospho-p38 MAPK (p-p38), p38 MAPK (p38), phospho-ERK1/2 (p-ERK1/2), and ERK1/2. GAPDH was used as an internal control. (B) Neutrophils were pretreated with inhibitor of p38 MAPK (SB203580) and ERK1/2 (U0126), and then cells were incubated with SS2 ZY05719 or PMA for 3 h. Results were normalized to neutrophils without pretreatment and activation, and are depicted as the mean ±SEM ( n = 3) of three independent experiments; ** p < 0.01; *** p < 0.001. (C) Neutrophils were pretreated with inhibitor of p38 MAPK (SB203580) and ERK1/2 (U0126), and the cells were then incubated with either SS2 ZY05719 or PMA for 3 h. Immunofluorescence was performed using anti-neutrophil elastase (NE) antibody followed by goat anti-rabbit Alexa 568 antibody (red). DNA was stained with DAPI (blue). The results shown are representative of three independent experiments.

Article Snippet: Thereafter, the membranes were incubated with Phospho-p38 MAPK (Thr180/Thr182) (D3F9) XP ® Rabbit mAb, p38 MAPK (D13E1) XP® Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)XP ® Rabbit mAb, p44/42 MAPK (Erk1/2)(137F5) Rabbit mAb, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (1:1000, Cell Signaling Technology, Danvers, MA, USA), or anti-GAPDH Rabbit pAb (1:5000, CMCTAG, Milwaukee, WI, USA) at 4°C overnight.

Techniques: Activation Assay, Infection, Western Blot, Incubation, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Streptococcus Suis Serotype 2 Stimulates Neutrophil Extracellular Traps Formation via Activation of p38 MAPK and ERK1/2

doi: 10.3389/fimmu.2018.02854

Figure Lengend Snippet: Western blotting analysis of phosphorylation of p38 MAPK and ERK1/2. (A) Neutrophils were pretreated with inhibitors of TLR4 signaling (TAK-242) and NADPH oxidase (DPI) for 30 min, and the cells were then incubated with SS2 ZY05719 and PMA for 2 h. Phosphorylation of p38 MAPK (p-p38) and ERK1/2 (p-ERK1/2) were determined with western blotting. (B) Neutrophils isolated form wild-type mice and TLR knockout mice were incubated with SS2 ZY05719 and Medium.

Article Snippet: Thereafter, the membranes were incubated with Phospho-p38 MAPK (Thr180/Thr182) (D3F9) XP ® Rabbit mAb, p38 MAPK (D13E1) XP® Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)XP ® Rabbit mAb, p44/42 MAPK (Erk1/2)(137F5) Rabbit mAb, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (1:1000, Cell Signaling Technology, Danvers, MA, USA), or anti-GAPDH Rabbit pAb (1:5000, CMCTAG, Milwaukee, WI, USA) at 4°C overnight.

Techniques: Western Blot, Incubation, Isolation, Knock-Out

Journal: International Journal of Molecular Medicine

Article Title: Hepatocyte growth factor inhibits hypoxia/reoxygenation-induced activation of xanthine oxidase in endothelial cells through the JAK2 signaling pathway

doi: 10.3892/ijmm.2016.2708

Figure Lengend Snippet: PI3K and JAK2 signaling pathways are involved in the production and activation of xanthine oxidase (XO). (A) Representative western blot of XO and tubulin expression in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). LY294002 and AG490 significantly decreased the increase in the XO protein level induced by H/R. (B) Summary data (n=3 biological replicates) of western blot analysis of XO and tubulin expression in CMECs following H/R. LY294002, SB203580 and AG490 treatment group compared with H/R group. * p<0.05 vs. H/R group. PI3K and JAK2 signaling pathways are involved in the production and activation of xanthine oxidase (XO). (C) Representative western blots of phosphorylated (p-)PI3K, total (t-)PI3K, p-JAK2, t-JAK2, p-p38 MAPK and t-p38 MAPK expression in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). H/R activates the PI3K and JAK2 signaling pathways but not the p38 MAPK signaling pathway. (D) Summary data (n=3 biological replicates) of western blot analysis of p-PI3K, t-PI3K, p-JAK2, t-JAK2, p-p38 MAPK and t-p38 MAPK expression in CMECs. ** p<0.01 vs. control group. (E) Representative data for flow cytometric analysis of DCFH-DA-stained CMECs following H/R. LY294002, SB203580 and AG490 treatment groups compared with H/R group. (F) Summary data (n=3 biological replicates) of flow cytometric analysis of DCFH-DA-stained CMECs following H/R. LY294002 (1 µ M), SB203580 (30 µ M) and AG490 (30 µ M) treatment groups compared with H/R group. * p<0.05 vs. H/R group; ** p<0.01 vs. H/R group.

Article Snippet: Forty micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and then probed with antibodies for XO (#ab109235; Abcam), phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199) (#4228), PI3 kinase p85 (19H8) (#4257), phospho-p38 MAP kinase (Thr180/Thr182) (#4631), p38 MAPK (D13E1) (#8690), JAK2 (D2E12) (#3230) and phospho-JAK2 (Tyr1007/1008) (C80C3) (#3776) (all from Cell Signaling Technology, Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Staining

Journal: International Journal of Molecular Medicine

Article Title: Hepatocyte growth factor inhibits hypoxia/reoxygenation-induced activation of xanthine oxidase in endothelial cells through the JAK2 signaling pathway

doi: 10.3892/ijmm.2016.2708

Figure Lengend Snippet: PI3K siRNA and JAK2 siRNA downregulate the production and activation of xanthine oxidase (XO). (A) Representative western blots of p38 MAPK, PI3K, JAK2 and tubulin expression in cardiac microvascular endothelial cells (CMECs). (B) Representative western blot of XO and tubulin expression in CMECs following hypoxia/reoxygenation (H/R). PI3K siRNA-, p38 MAPK siRNA-, JAK2 siRNA- and negative control siRNA-transfected groups compared with H/R group. (C) Summary data (n=3 biological replicates) for western blot analysis of XO and tubulin expression in CMECs following H/R. PI3K siRNA-, p38 MAPK siRNA-, JAK2 siRNA- or negative control siRNA-transfected groups compared with H/R group. ** p<0.01 vs. H/R group. (D) Representative flow cytometric analysis of DCFH-DA-stained CMECs after H/R. PI3K siRNA-, p38 MAPK siRNA-, JAK2 siRNA- or negative control siRNA-transfected groups compared with H/R group. (E) Summary data (n=3 biological replicates) for flow cytometric analysis of DCFH-DA-stained CMECs after H/R. PI3K siRNA-, p38 MAPK siRNA-, JAK2 siRNA- or negative control siRNA-transfected groups compared with H/R group. ** p<0.01 vs. H/R group.

Article Snippet: Forty micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and then probed with antibodies for XO (#ab109235; Abcam), phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199) (#4228), PI3 kinase p85 (19H8) (#4257), phospho-p38 MAP kinase (Thr180/Thr182) (#4631), p38 MAPK (D13E1) (#8690), JAK2 (D2E12) (#3230) and phospho-JAK2 (Tyr1007/1008) (C80C3) (#3776) (all from Cell Signaling Technology, Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Negative Control, Transfection, Staining