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

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: Characterization of the senescent CD4 + T lymphocytes. ( a ) Flow cytometry gating strategy used to determine the purity of viable CD4 + T lymphocytes isolated from the spleen of wild-type C57 BL/6 mice. The following sequential gating strategy steps were used: First, cells were physically selected according to their cell size (FSC-A) and internal cell complexity (SSC-A) parameters. Second, only single cells were selected according to their FSC-H and FSC-A characteristics, discarding fused cells as duplets or triplets. Third, dead cells were excluded. Finally, the viable CD4 + T cells were selected for analysis. This cell characterization was performed separately for each animal. (b) Flow cytometry histogram and bar plot show the cell size quantification of viable CD4 + T lymphocytes exposed or non-exposed to oxidative stress (H 2 O 2 ). (c) Flow cytometry histogram and bar plot show the frequency of proliferating CD4 + lymphocytes. H 2 O 2 -exposed CD4 + T cells stimulated with CD3ε-CD28 (red) were compared with non-exposed CD4 + T cells stimulated with CD3ε-CD28 (gray). Non-exposed CD4 + T cells without CD3ε-CD28 stimulation were used as negative control (yellow). The dotted line indicates the reference point used to quantify the proliferating cells. (d) Relative quantification of the mRNA expression of the cell cycle inhibitors p16 Ink4a and p21 Cip1 in H 2 O 2 -exposed or non-exposed CD4 + T cells determined by RT-qPCR. (e) Western blot immunodetection of phospho-p38, total p38, and β-actin and quantification of the phospho-p38/total p38 band density in senescent and non-senescent CD4 + T lymphocytes. (f) Quantification of total p38/β-actin band density in senescent and non-senescent CD4 + T lymphocytes. Data are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The membrane was incubated with the following primary antibodies: rabbit anti-mouse-phospho-p38 (Cell Signaling Technology), rabbit anti-mouse-p38 (Abcam), and rabbit anti-mouse-β-actin (Santa Cruz Biotechnology) 1:2000, overnight at 4 °C.

Techniques: Flow Cytometry, Isolation, Negative Control, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Immunodetection

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: p38 MAPK in dysfunctional mitochondria accumulation and autophagy disruption in senescent CD4 + T lymphocytes. ( a ) Flow cytometry histogram and bar plot showing the MitoTracker dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. Non-senescent CD4 + T lymphocytes were used for comparison. (b) Relative quantification of the mRNA expression of the fusion/fission markers MFN2 and DRP1 in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO determined by RT-qPCR. (c) Flow cytometry histogram and bar plot showing the MitoTracker dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796, BIRB-796 plus Bafilomycin (autophagy inhibitor), Rapamycin (autophagy inducer), or DMSO. The histogram blue peak corresponds to senescent CD4 + T lymphocytes exposed to BIRB-796, the pink peak to senescent CD4 + T lymphocytes exposed to BIRB-796 plus Bafilomycin, the grey peak to senescent CD4 + T lymphocytes exposed to Rapamycin, the red peak to senescent CD4 + T lymphocytes exposed to DMSO, and the yellow peak to the negative control (MitoTracker dye MFO). (d) The same analysis described in (c) using the frequency of MitoTracker high senescent CD4 + T lymphocytes. (e) Flow cytometry histogram and bar plot showing the frequency of MitoSox-positive cells and MitoSox dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. Non-senescent CD4 + T lymphocytes were used for comparison. (f) Flow cytometry histogram and bar plot showing the H2DCFDA dye MFI in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. Non-senescent CD4 + T lymphocytes were used for comparison. In all flow cytometry histograms, the blue peak corresponds to senescent CD4 + T lymphocytes exposed to BIRB-796, the red peak to senescent CD4 + T lymphocytes exposed to DMSO, the grey peak to non-senescent CD4 + T lymphocytes, and the yellow peak to the negative controls (MitoTracker, MitoSox, and H2DCFDA dye MFO, respectively). Data are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student’s t-test, when comparing two groups, or the one-way ANOVA and Tukey post hoc tests, when comparing more than two experimental groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Baf, bafilomycin; DMSO, dimethyl sulfoxide; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate reagents; MFI, mean fluorescence intensity, MFO: fluorescence minus one; Rap, rapamycin

Article Snippet: The membrane was incubated with the following primary antibodies: rabbit anti-mouse-phospho-p38 (Cell Signaling Technology), rabbit anti-mouse-p38 (Abcam), and rabbit anti-mouse-β-actin (Santa Cruz Biotechnology) 1:2000, overnight at 4 °C.

Techniques: Disruption, Flow Cytometry, Comparison, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Negative Control, Fluorescence

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: Role of p38 MAPK in the Th17-type SASP release in senescent CD4 + T lymphocytes. ( a ) Quantification of the secreted levels of IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-23, and GM-CSF in senescent and non-senescent CD4 + T lymphocytes determined by ELISA-Multiplex. (b) Relative quantification of the mRNA expression of RORγt, Tbx 21, STAT-4, and CCL4 in senescent and non-senescent CD4 + T lymphocytes determined by RT-qPCR. (c) Quantification of the secreted levels of IL-6, IL-17A, and GM-CSF in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. (d) Relative quantification of the mRNA expression of RORγt and Tbx 21 in senescent CD4 + T lymphocytes exposed to BIRB-796 or DMSO. (e) Flow cytometry and bar plots showing the number and frequency of senescent CD4 + T lymphocytes exposed to BIRB-796, exposed to DMSO, and non-senescent CD4 + T lymphocytes expressing IL-17A (CD4 + IL-17A + T cells). Data are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student’s t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. CCL, chemokine ligand; DMSO, dimethyl sulfoxide; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; ROR, retinoid-related orphan receptor; SASP, senescence-associated secretory phenotype; STAT, signal transducer and activator of transcription; Tbx, T-box transcription factor

Article Snippet: The membrane was incubated with the following primary antibodies: rabbit anti-mouse-phospho-p38 (Cell Signaling Technology), rabbit anti-mouse-p38 (Abcam), and rabbit anti-mouse-β-actin (Santa Cruz Biotechnology) 1:2000, overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Multiplex Assay, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Immunity & Ageing : I & A

Article Title: p38 mitogen-activated protein kinase drives senescence in CD4 + T lymphocytes and increases their pathological potential

doi: 10.1186/s12979-025-00526-8

Figure Lengend Snippet: Graphic summary that contextualizes the main findings of the present study. Activation of p38 MAPK drives cellular senescence in CD4 + T lymphocytes. Particularly, p38 MAPK activation causes mitophagy disruption, accumulation of dysfunctional mitochondria, and production of an SASP enriched in Th17-type mediators. These cell-senescent phenomena were inhibited when BIRB-796 was used to neutralize the p38 MAPK activation

Article Snippet: The membrane was incubated with the following primary antibodies: rabbit anti-mouse-phospho-p38 (Cell Signaling Technology), rabbit anti-mouse-p38 (Abcam), and rabbit anti-mouse-β-actin (Santa Cruz Biotechnology) 1:2000, overnight at 4 °C.

Techniques: Activation Assay, Disruption

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