rabbit anti phospho p38mapk  (Cell Signaling Technology Inc)

Journal: Translational Lung Cancer Research

Article Title: Targeting of arachidonic acid-modulated autophagy to enhance the sensitivity of ROS1 + or ALK + non-small cell lung cancer to crizotinib therapy

doi: 10.21037/tlcr-2025-105

Figure Lengend Snippet: Downregulation of MAPK/p-STAT3/PTGS2 drove metabolic reprogramming in crizotinib-resistant cells. (A) UMAP plots based on the top 5 principal components of all single-cell transcriptomes after quality control, color-coded by treatment group (ALK TKI-sensitive or TKI-resistant) or by subsets identified through unsupervised dimensionality reduction and clustering. The analysis revealed two TKI-sensitive clusters (SensC1 and SensC2) and six TKI-resistant clusters (ResiC1–ResiC6). (B) Differentially expressed genes in each subset, with the top 5 genes per subset being shown (see A for color codes). (C) Mean pathway activity scores for different cell subsets. (D) Human phospho-kinase array analysis was performed to evaluate signaling pathways in HCC78 and HCC78CR cells treated with 2 µM of crizotinib for 24 hours. The green arrow indicates the puncta of p-STAT3 (S727). (E) HCC78 and HCC78CR cells were treated with the 1 µM crizotinib for 24 hours. The protein levels of p-P38MAPK (T180/T182) and p-STAT3 (S727) were detected via western blotting. β-actin was used as the loading control. The gray value ratios of phosphorylated to total proteins are shown on the right. (F) Volcano plot of RNA-sequencing analysis comparing gene expression profiles between parental and resistant HCC78 cells. (G) KEGG pathway enrichment analysis of differentially expressed genes between parental and resistant HCC78 cells. (H) CNET mapping of four differential signaling pathways (lL-17 signaling pathway, cytokine-cytokine receptor interaction, NF-κB signaling pathway, and regulation of lipolysis in adipocytes). (I) UMAP and dot plots showing the expression levels of EPCAM and PTGS2 in each subset in ALK-TKI-sensitive and TKI-resistant tumors. (J) Unsupervised transcriptional trajectory analysis of different cell subsets generated using R package Monocle2, colored by pseudotime, cell subsets, and PTGS2 expression levels, respectively, in ALK TKI-sensitive and TKI-resistant tumors. (K) The levels of PTGS2 mRNA in HCC78CR and H3122CR cells, as compared to their corresponding parental cells, were measured using RT-qPCR. Data are presented as the mean ± SD. (E) Brown-Forsythe and Welch ANOVA tests and (K) Unpaired two-sided Student’s t -test. ns: no significance. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance; ALK, anaplastic lymphoma kinase; Cri, crizotinib; CNET, computational network; CR, crizotinib resistance; EPCAM, epithelial cell adhesion molecule; KEGG, Kyoto Encyclopedia of Genes and Genomes; p38MAPK, mitogen-activated protein kinase; p-p38MAPK(T180/182), phosphorylation of mitogen-activated protein kinase on T180 and T182; STAT3, signal transducer and activator of transcription 3; p-STAT3(S727), phosphorylation of signal transducer and activator of transcription 3 on S727; no-diff, no difference; PTGS2, prostaglandin endoperoxide synthase 2; SD, standard deviation; TKI, tyrosine kinase inhibitor; UMAP, uniform manifold approximation and projection; WT, wild type.

Article Snippet: The primary antibodies used were LAMP1 [cat. no. 9091; Cell Signaling Technology (CST), Danvers, MA, USA], LC3B (cat. no. 83506; CST), p-p38MAPK (Thr180/Tyr182) (cat. no. 4511; CST), p38 MAPK (cat. no. 8690; CST), p-STAT3 (S727) (cat. no. 9134; CST), STAT3 (cat. no. 9139; CST), p-IRE1 (S724) (cat no. AP1442; ABclonal, Woburn, MA, USA), IRE1 (cat no. A17940; ABclonal), sXBP1 (cat no. A17007; ABclonal), p-PERK (T982) (cat no. AP0886; ABclonal), and PERK (cat. no. A18196; ABclonal). β-actin, HRP-conjugated goat anti-rabbit, and goat anti-mouse secondary antibodies were obtained from Boster Bio (Pleasanton, CA, USA).

Techniques: Control, Activity Assay, Protein-Protein interactions, Western Blot, RNA Sequencing, Gene Expression, Expressing, Generated, Quantitative RT-PCR, Phospho-proteomics, Standard Deviation

Journal: Biomolecules & therapeutics

Article Title: Interleukin-1β Signaling Contributes to Cell Cycle Arrest and Apoptotic Cell Death by Leptin via Modulation of AKT and p38MAPK in Hepatocytes.

doi: 10.4062/biomolther.2023.232

Figure Lengend Snippet: Fig. 5. Role of p38MAPK activation in apoptosis and cell cycle arrest by leptin in rat hepatocytes. (A) Hepatocytes were treated with leptin (250 ng/mL) for the indicated time and phosphor-p38MAPK levels were analyzed by western blot analysis. (B-E) Hepatocytes were pre- treated with SB203580 (10 µM), a pharmacological inhibitor of p38MAPK, for 1 h followed by incubation with leptin (250 ng/mL) for addition- al 24 h (B, D) or 8 h (C, E). (B) Cells were stained with annexin V/7-AAD and apoptotic cell death was determined by flow cytometry analy- sis. Cells in each quadrant were quantified and presented in the right panel. (C) Expression levels of cleaved caspase-3 were measured by western blot analysis. (D) Cell cycle analysis was performed by PI staining and flow cytometry analysis. Cells in each phase of cell cycle is quantified and presented in the right panel. (E) Expression levels of p16 were measured by western blot analysis. (F-H) Hepatocytes were pretreated with SC79 (4 µg/mL) (F), SB216763 (5 µM) (G), or IL-1Ra (100 ng/mL) (H) for 1 h followed by incubation with leptin (250 ng/mL) for additional 8 h. Phosphor-p38MAPK levels were determined by western blot analysis. Representative images from at least three indepen- dent sets of experiment is shown for both flow cytometry and western blot analysis. In western blot analysis, the band intensities of the genes of interest were quantified by densitometric analysis and presented in the lower panel. β-actin or total form of corresponding protein was used as internal control. Values are presented as mean ± SEM, n=3. *p<0.05 in comparison to control cells, #p<0.05 in comparison to the cells treated with leptin.

Article Snippet: Primary antibodies against Bax (2772), Bcl-2 (2876), pro-caspase-3 (9662), cleaved caspase-3 (9664), PARP (9542), phosphor-AKT (4060S), phosphorGSK3β (5558S), and phosphor-p38MAPK (9215) were acquired from Cell Signaling Technologies (Danvers, CO, USA).

Techniques: Activation Assay, Western Blot, Incubation, Staining, Flow Cytometry, Expressing, Cell Cycle Assay, Control, Comparison

Journal: Biomolecules & therapeutics

Article Title: Interleukin-1β Signaling Contributes to Cell Cycle Arrest and Apoptotic Cell Death by Leptin via Modulation of AKT and p38MAPK in Hepatocytes.

doi: 10.4062/biomolther.2023.232

Figure Lengend Snippet: Fig. 6. Effects of leptin on apoptosis, p16 expression, and modulation of AKT/p38MAPK pathways in hepatocyte specific IL-1R1 knockout mice liver. (A) Hepatocyte specific IL-1R1 knockout mice was generated using loxP/Cre system. Messenger RNA levels of the correspond- ing genes were meaured by PCR amplification as described in the methods. In left panel, lane-1: IL-1R1 fl/fl mice, lane-2: heterozygous for IL-1R1, lane-3: WT for IL-1R1, lane-4: IL-1R1 fl/fl mice. In right panel, lanes 1 and 2: Cre recombinase detected, lanes 3 rd and 4 th: no expres- sion of Cre recombinase. (B, C) Expression levels of IL-1R1 in liver (B) and hepatocytes (C) were measured by western blot analysis. (D-I) Mice were adminstered with leptin (1 mg/kg) intraperitoneally twice a day for 15 days. After treatment, livers were prepared as indicated in the methods and expression levels of cleaved caspase-3 (D), cleaved cytokeratin-18 (E), Bax (F), Bcl-2 (G), p16 (H), phosphor-AKT (I), and phosphor-p38MAPK (J) were measured by western blot analysis. Representative images from three mice out of total five mice in each group is presented. Values are presented as mean ± SEM, n=5. * denotes p<0.05 in comparison to WT-control. # denotes p<0.05 in com- parison to WT-leptin.

Article Snippet: Primary antibodies against Bax (2772), Bcl-2 (2876), pro-caspase-3 (9662), cleaved caspase-3 (9664), PARP (9542), phosphor-AKT (4060S), phosphorGSK3β (5558S), and phosphor-p38MAPK (9215) were acquired from Cell Signaling Technologies (Danvers, CO, USA).

Techniques: Expressing, Knock-Out, Generated, Amplification, Western Blot, Comparison, Control

Journal: Biomolecules & therapeutics

Article Title: Interleukin-1β Signaling Contributes to Cell Cycle Arrest and Apoptotic Cell Death by Leptin via Modulation of AKT and p38MAPK in Hepatocytes.

doi: 10.4062/biomolther.2023.232

Figure Lengend Snippet: Fig. 7. Proposed model for the role of interleukin-1β signaling in leptin-induced hepatocyte death. Leptin induces maturation of interleukin- 1β (IL-1β) through NLRP3 inflammasomes signaling, which itself was found to be activated via ER stress-dependent mechanism in our pre- vious report (Baral and Park, 2021). Once IL-1β is activated, it is secreted into the extracellular milleu, where it binds to the type-I interleu- kin-1 receptor (IL-1R1) and transmits the signal that induce apoptosis and cell cycle arrest in hepatocytes. Mechanistically, conformational change of IL-1R1 upon binding with IL-1β leads to inactivation of AKT, which plays a role in the survival and proliferation of hepatocyte. In- activation of AKT causes dephosphorylation (at Ser 9) and subsequent activation of GSK3β, further resulting in activation of the stress ki- nase p38MAPK. Finally, p38MAPK signaling critically contributes to p16-dependent cell cycle arrest and apoptotic cell death. Illustration created with BioRender.com.

Article Snippet: Primary antibodies against Bax (2772), Bcl-2 (2876), pro-caspase-3 (9662), cleaved caspase-3 (9664), PARP (9542), phosphor-AKT (4060S), phosphorGSK3β (5558S), and phosphor-p38MAPK (9215) were acquired from Cell Signaling Technologies (Danvers, CO, USA).

Techniques: Binding Assay, Activation Assay, De-Phosphorylation Assay