rabbit anti-mouse phospho-p38-mapk  (Cell Signaling Technology Inc)

Journal: Blood Advances

Article Title: Neuroprotective, anti-inflammatory, and analgesic activity of palmitoylethanolamide in sickle cell mice

doi: 10.1182/bloodadvances.2024015439

Figure Lengend Snippet: Inhibition of p38-MAPK ameliorates chronic hyperalgesia in HbSS mice. (A) Western immunoblotting revealed decreased p38-MAPK phosphorylation in the spinal cord of HbSS mice treated with PEA (IP, 14 days, 20 mg/kg per day). (B) PEA (30 μM) reduced phosphorylation of p38-MAPK and nuclear translocation of phosphorylated p38-MAPK in primary DRG neurons collected from female HbSS mice. Representative images of DRG neurons treated with Veh (complete media), TNF-α (1 ng/mL)/hemin (40 μM), and/or PEA (30 μM) immunolabeled with primary rabbit anti-mouse phospho-p38-MAPK (1:100; catalog no. 9211S, Cell Signaling Technology), secondary Cy3 AffiniPure donkey anti-rabbit immunoglobulin G (H+L), Absorption max: 550 nm and Excitation max: 570 nm (1:500; catalog no. 711-165-152, Jackson ImmunoResearch, West Grove, PA) antibodies, and DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain (1:25 000; catalog no. D1306, Invitrogen, Thermo Fisher). (B-C) DRG neurons showing p38-MAPK phosphorylation and phospho-p38-MAPK nuclear colocalization were enumerated and averaged in 6 fields of view per subject. Incitement of a sickle microenvironment significantly increased phospho-p38-MAPK nuclear colocalization, which was completely attenuated with PEA cotreatment. Z-stacks of 10× 0.5-μm images were acquired on a laser scanning confocal microscope (Zeiss LSM 900, Carl Zeiss AG) using a plan-apochromat 63× oil M27 objective lens. (D-G) Targeting p38-MAPK with Nef dose-dependently (oral 6 or 12 mg/kg twice daily) reduced PWF in response to mechanical and cold stimuli and reduced cold aversion at days 7 and 14 of treatment, without affecting grip force in female HbSS mice, suggesting reduced hyperalgesia. Mean ± SD. In panels A-C, data were analyzed with unpaired Student 2-tailed t test. In panels D-G, data were analyzed with 2-way ANOVA and the Tukey post hoc multiple comparisons test. ∗Indicates difference compared with Veh; †indicates difference compared with corresponding BL. ∗,† P < .05; ∗∗,†† P < .01; ∗∗∗,††† P < .001; ∗∗∗∗ P < .0001. Age: 3.5 to 5.0 months. For panels A-C, n = 3 per condition; panels D-G; female HbSS Veh, n = 5; female HbSS Nef 6 mg/kg, n = 5; female HbSS Nef 12 mg/kg, n = 5; female HbAA Veh, n = 6; female HbAA Nef 12 mg/kg, n = 6. BW, body weight; IR, immunoreactivity; PWF, paw withdrawal frequency; Temp, temperature; VF, von Frey.

Article Snippet: Representative images of DRG neurons treated with Veh (complete media), TNF-α (1 ng/mL)/hemin (40 μM), and/or PEA (30 μM) immunolabeled with primary rabbit anti-mouse phospho-p38-MAPK (1:100; catalog no. 9211S, Cell Signaling Technology), secondary Cy3 AffiniPure donkey anti-rabbit immunoglobulin G (H+L), Absorption max: 550 nm and Excitation max: 570 nm (1:500; catalog no. 711-165-152, Jackson ImmunoResearch, West Grove, PA) antibodies, and DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain (1:25 000; catalog no. D1306, Invitrogen, Thermo Fisher). (B-C) DRG neurons showing p38-MAPK phosphorylation and phospho-p38-MAPK nuclear colocalization were enumerated and averaged in 6 fields of view per subject.

Techniques: Inhibition, Western Blot, Phospho-proteomics, Translocation Assay, Immunolabeling, Microscopy

Journal: Antioxidants

Article Title: Neuronal p38 MAPK Signaling Contributes to Cisplatin-Induced Peripheral Neuropathy

doi: 10.3390/antiox14040445

Figure Lengend Snippet: Cisplatin-induced p38 MAPK activation in DRG neurons. ( A – C ). Effect of age, genotype, and dose on p38 MAPK phosphorylation and nuclear translocation in the cisplatin-treated DRG neurons of FVB/N and C3TAg female mice. ( D – F ). Neflamapimod 5 µM inhibits cisplatin-induced p38 MAPK phosphorylation and nuclear translocation in primary DRG neurons isolated from female FVB/N and C3TAg mice. Images of primary DRG neurons in vitro show co-expression of phospho-p38 MAPK-immunoreactivity (ir, red) and cell nuclei (DAPI, cyan). The yellow arrow indicates phospho-p38 MAPK-ir nuclei. Confocal microscope 63×/NA 1.4 oil was used to capture DRG neurons from 3–6 mice per group. The percentage of phospho-p38 MAPK-ir nuclei and fluorescence was averaged from 6 random fields per group. Data are shown as the mean ± SEM and analyzed with 2-way ANOVA and Tukey’s multiple comparisons test. Abbreviations: DRG, dorsal root ganglia; ir, immunoreactivity; p38 MAPK, P38 mitogen-activated protein kinases; Phospho, phosphorylated.

Article Snippet: Fixed cells were permeabilized with ice-chilled 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 2 min, blocked with 3% donkey serum in PBS for 30 min, and incubated overnight at 4 °C with rabbit anti-mouse phospho-p38 MAPK antibody (#9211S; Cell Signalling Technology [CST], Danvers, MA, USA; 1:100), or rabbit anti-mouse cleaved caspase 3 antibody (#9661; CST, Danvers, MA, USA; 1:200) or rabbit anti-β3-Tubulin (#5568; CST, Danvers, MA, USA; 1:200) diluted in blocking buffer (3% donkey serum in PBS), followed by secondary antibodies, Cy TM 3 AffiniPure Donkey Anti-Rabbit IgG (H+L), Amax: 550 Emax: 570 nm (#711-165-152, Jackson ImmunoResearch, West Grove, PA, USA), (1:500 dilution in blocking buffer) for 1 h at RT; and mounted with ProLong TM diamond antifade mounting media (Thermo Fisher Scientific, Waltham, MA, USA) containing nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI).

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay, Isolation, In Vitro, Expressing, Microscopy, Fluorescence

Journal: Antioxidants

Article Title: Neuronal p38 MAPK Signaling Contributes to Cisplatin-Induced Peripheral Neuropathy

doi: 10.3390/antiox14040445

Figure Lengend Snippet: In vivo neflamapimod inhibits cisplatin-induced phopho-p38 MAPK activation and colocalization in nuclei of DRG neurons. C3TAg and FVB/N mice were treated with vehicle, neflamapimod, and cisplatin ± neflamapimod as described under the Methods. DRG neurons were isolated from all groups of mice after treatments. ( A ) Images of primary DRG neurons show the co-expression of phospho-p38 MAPK-immunoreactivity (ir, red) and cell nuclei (DAPI, cyan). Yellow arrows indicate the phospho-p38 MAPK-ir nuclei. Confocal microscope magnification: 63×/1.4 oil. Z-stacks of 0.5 µm images from 6 fields of view were captured for each condition, from DRG neurons obtained from 4–6 mice per condition. ( B , C ) The percentage of phospho-p38 MAPK-ir nuclei and fluorescence was averaged from 6 randomly selected fields per group. Data are shown as the mean ± SEM analyzed with 2-way ANOVA and Tukey’s multiple comparisons test. Abbreviations: ir, immunoreactivity; p38 MAPK, P38 mitogen-activated protein kinase; Phospho, phosphorylated.

Article Snippet: Fixed cells were permeabilized with ice-chilled 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 2 min, blocked with 3% donkey serum in PBS for 30 min, and incubated overnight at 4 °C with rabbit anti-mouse phospho-p38 MAPK antibody (#9211S; Cell Signalling Technology [CST], Danvers, MA, USA; 1:100), or rabbit anti-mouse cleaved caspase 3 antibody (#9661; CST, Danvers, MA, USA; 1:200) or rabbit anti-β3-Tubulin (#5568; CST, Danvers, MA, USA; 1:200) diluted in blocking buffer (3% donkey serum in PBS), followed by secondary antibodies, Cy TM 3 AffiniPure Donkey Anti-Rabbit IgG (H+L), Amax: 550 Emax: 570 nm (#711-165-152, Jackson ImmunoResearch, West Grove, PA, USA), (1:500 dilution in blocking buffer) for 1 h at RT; and mounted with ProLong TM diamond antifade mounting media (Thermo Fisher Scientific, Waltham, MA, USA) containing nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI).

Techniques: In Vivo, Activation Assay, Isolation, Expressing, Microscopy, Fluorescence

Journal: Antioxidants

Article Title: Neuronal p38 MAPK Signaling Contributes to Cisplatin-Induced Peripheral Neuropathy

doi: 10.3390/antiox14040445

Figure Lengend Snippet: Cisplatin-induced hyperalgesia is mitigated by a p38 MAPK inhibitor neflamapimod. Cisplatin treatment activates p38 MAPK in DRG neurons of C3TAg mice with mammary tumors. Neflamapimod, a p38α MAPK inhibitor, inhibits this activation and reduces cisplatin-induced oxidative stress, mitochondrial dysfunction, and apoptosis. It preserved neuronal integrity and axonal structure, while ameliorating cisplatin-induced hyperalgesia. The green upward arrow represents an increase in hyperalgesia following cisplatin treatment, while the red downward arrow indicates a reduction with neflamapimod co-treatment. Abbreviations: DRG, dorsal root ganglia; OG, oral gavage; p38 MAPK, p38 Mitogen-activated protein kinase.

Article Snippet: Fixed cells were permeabilized with ice-chilled 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 2 min, blocked with 3% donkey serum in PBS for 30 min, and incubated overnight at 4 °C with rabbit anti-mouse phospho-p38 MAPK antibody (#9211S; Cell Signalling Technology [CST], Danvers, MA, USA; 1:100), or rabbit anti-mouse cleaved caspase 3 antibody (#9661; CST, Danvers, MA, USA; 1:200) or rabbit anti-β3-Tubulin (#5568; CST, Danvers, MA, USA; 1:200) diluted in blocking buffer (3% donkey serum in PBS), followed by secondary antibodies, Cy TM 3 AffiniPure Donkey Anti-Rabbit IgG (H+L), Amax: 550 Emax: 570 nm (#711-165-152, Jackson ImmunoResearch, West Grove, PA, USA), (1:500 dilution in blocking buffer) for 1 h at RT; and mounted with ProLong TM diamond antifade mounting media (Thermo Fisher Scientific, Waltham, MA, USA) containing nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI).

Techniques: Activation Assay

Journal: International journal of molecular sciences

Article Title: WGA-M001, a Mixture of Total Extracts of Tagetes erecta and Ocimum basilicum , Synergistically Alleviates Cartilage Destruction by Inhibiting ERK and NF-κB Signaling.

doi: 10.3390/ijms242417459

Figure Lengend Snippet: Figure 4. WGA-M001 regulated OA-related molecules through dephosphorylation in the NF-κB and ERK pathways. For in silico analysis, 200 µg/mL of Tagetes erecta, Ocimum basilicum, or WGA-M001 was used for treatment of chondrocytes for 12 h along with IL-1β (1 ng/mL), and RNA sequencing was performed. (A) In each signaling pathway, the number of genes upregulated by IL-1β and then downregulated by T. erecta, O. basilicum, or WGA-M001 was shown. (B,C) Protein levels of pp38, p38, pJNK, JNK, pERK, ERK, pp65, p65, and IκB were detected by Western blot analysis and relative intensities were quantified by densitometry (n = 5). Each protein level was normalized to ERK. Data are represented as mean ± SD as results of analysis by one-way ANOVA with Dunnett’s multiple comparisons test (n = 5). * p < 0.05, **** p < 0.0001, and n.s = not significant.

Article Snippet: 2023, 24, 17459 12 of 16 cam), rabbit anti-COX-2 (ab52237; Abcam), mouse anti-IκB (9242; Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-p65 (#6956; CST), mouse anti-pp65 (#13346; CST), mouse anti-p38 (#9212; CST), mouse anti-pp38 (#9215S; CST), mouse anti-c-JNK (#9252S; CST), mouse anti-pJNK (#9251S; CST), and mouse anti-pERK (#9101S; CST).

Techniques: De-Phosphorylation Assay, In Silico, RNA Sequencing, Western Blot