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: PLoS ONE

Article Title: Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects

doi: 10.1371/journal.pone.0031857

Figure Lengend Snippet: A+B: Identification of the Epo-R in skeletal muscle tissue in the non-stimulated state in all 10 subjects with the C20 (A) and M20 (B) antibodies. Positive control is the k-562 cells. M is the molecular marker. C: Activation of the Epo-R by phosphorylation evaluated by western blotting. The samples from the same subject in the stimulated and un-stimulated situation are loaded next to each other D: Phosphorylation of Epo-R, STAT5, p38-MAPK, Akt, p70S6 kinase, Lyn, and IKK, all normalized to the levels of the given protein. The mRNA levels of SOCS3 and IGF-I are normalized to beta-actin mRNA content. Black bars: placebo, white bars: rHuEpo. All results are from biopsies taken 1 h post treatment with either placebo or rHuEpo.

Article Snippet: The protein fraction was analysed for phosphorylation of Epo-R, Lyn, STAT5, Akt, p70S6-kinase, MAPK and for total Epo-R. Primary antibodies were as follows: anti-phospho-Epo-R(Tyr456) (Santa Cruz, #sc20236), anti-Lyn (Cell signalling, #2732), anti-phospho-Lyn(Tyr507) (Cell signalling, #2731), anti-STAT5 (Cell signalling, #9310), anti-phospho-STAT5(Tyr694) (Cell signalling, #9351), anti-Akt/PKB (Cell signalling, #9272), anti-phospho-Akt/PKB(Ser473) (Cell signalling, #9271), anti-phospho-Akt/PKB(Thr308) (Cell signalling, #9275), anti-p70S6 kinase (Cell signalling, #9202), anti-phospho-p70S6 kinase(Thr389) (Cell signalling, #9205), anti-p38-MAP kinase (Cell signalling, #9212), anti-phospho-p38-MAP kinase (Thr180/Thr182) (Cell signalling, #9211), anti-IKKα (Cell signalling, #2682), anti-phospho-IKKα/β(Ser176/180) (Cell signalling, #2697), anti-Epo-R (M20) (Santa Cruz, #sc697), anti-Epo-R (C20) (Santa Cruz, #sc695), and anti-β-actin (Abcam, #ab8227).

Techniques: Positive Control, Marker, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects

doi: 10.1371/journal.pone.0031857

Figure Lengend Snippet: Phosphorylation of STAT5, p38-MAPK, Akt, p70S6 kinase, Lyn, and IKK, all normalized to the total levels of the given protein. Black bars: placebo, white bars: rHuEpo. The levels of mRNA are measured at 10 h post rHuEpo administration, and are normalized to beta-actin mRNA levels. Level of significance p<0.05, * compared to baseline, # compared to control (same timepoint), § compared to rHuEpo 2 h. The interactions were as follows; pEpo-R p = 0.318, p38-MAPK p = 0.058 (treatment effect p = 0.030), pSTAT5 p = 0.562, p-Akt(ser) p = 0.001, pAkt(thr) p = 0.007, p-IKK p = 0.742 (time effect p = 0.017), p-LYN p = 0.427, p-p70S6kinase p = 0.164 (time effect p = 0.033).

Article Snippet: The protein fraction was analysed for phosphorylation of Epo-R, Lyn, STAT5, Akt, p70S6-kinase, MAPK and for total Epo-R. Primary antibodies were as follows: anti-phospho-Epo-R(Tyr456) (Santa Cruz, #sc20236), anti-Lyn (Cell signalling, #2732), anti-phospho-Lyn(Tyr507) (Cell signalling, #2731), anti-STAT5 (Cell signalling, #9310), anti-phospho-STAT5(Tyr694) (Cell signalling, #9351), anti-Akt/PKB (Cell signalling, #9272), anti-phospho-Akt/PKB(Ser473) (Cell signalling, #9271), anti-phospho-Akt/PKB(Thr308) (Cell signalling, #9275), anti-p70S6 kinase (Cell signalling, #9202), anti-phospho-p70S6 kinase(Thr389) (Cell signalling, #9205), anti-p38-MAP kinase (Cell signalling, #9212), anti-phospho-p38-MAP kinase (Thr180/Thr182) (Cell signalling, #9211), anti-IKKα (Cell signalling, #2682), anti-phospho-IKKα/β(Ser176/180) (Cell signalling, #2697), anti-Epo-R (M20) (Santa Cruz, #sc697), anti-Epo-R (C20) (Santa Cruz, #sc695), and anti-β-actin (Abcam, #ab8227).

Techniques:

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