Phospho-JAK2 (Tyr931) Antibody - #AF3024

Figure 4 ITGA5-induced NGF secretion in Schwann cells (A) Representative figures of GFAP (red) and p75NTR (green) staining in tumor tissues. Scale bar: 20 μm. (B) Quantification of GFAP+ cells and GFAP+p75NTR+ cells. (C) Flow cytometry analysis of GFAP and c-Jun expression in Schwann cells (SCs). (D) Relative expression of Ngf mRNA. The y axis represents the relative expression levels using the housekeeping gene Gapdh as the standard. CCs, cancer cells. n = 3 technical replicates. (E) Quantification of secreted NGF in CCs and SCs by ELISA (n = 3 technical replicates). (F) Predicted interaction of fibronectin to protein itga5. Nodes represent individual proteins, with ITGA5/Itga5 as the central node. Edges indicate predicted interactions, with line thickness reflecting confidence scores. Line colors denote evidence types: curated databases (sky blue), experimental interactions (purple), co-expression (black), etc. Functional partners are labeled with gene symbols. (G) Molecular model of the interaction between ITGA5 (blue) and FN1 (red). At right is an enlarged view of interacting amino acid residues. (H) Expression levels of Fn1 mRNA in NC and OE groups. (I) Relative expression of Ngf mRNA. The y axis represents the relative expression levels using the housekeeping gene Gapdh as the standard. NC, negative control. (J) Quantification of secreted NGF in si-Ctrl and si-Fn1 SCs detected by ELISA (n = 3 technical replicates). (K) Transcript levels of reparative SC markers in samples from each group. (L) KEGG pathway enrichment analysis of genes significantly downregulated by si-Fn1 treatment. (M) Representative western blot images showing the expression of STAT3, p-STAT3, JAK2, p-JAK2, and GAPDH as the loading control. Quantitative analysis of band intensity normalized to GAPDH. CM, conditioned medium. Each bar represents the mean (SEM) from three independent experiments. ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical comparisons were performed using two-tailed independent samples t tests.

Fig. 3 The underlying mechanism by which aligned fibers affected macrophage polarization. A Venn diagram showing differentially expressed genes. B KEGG pathway analysis between the A20 and R20 groups. C Heatmap of differentially expressed genes among the three groups. D Heatmap of macrophage polarization-related genes between the A20 and R20 groups. E Volcano diagram of differentially expressed genes. F Western blot analysis of the NF-κB signaling pathway. G Immunofluorescence staining showing the nuclear translocation of NF-κB p65. The nucleus is stained blue, and NF-κB p65 protein is stained red. H Western blot images and semiquantitative analysis of the JAK-STAT signaling pathway (*p < 0.05, **p < 0.01, n = 3)

Figure 3. Oncogenic potential and altered molecular signaling in Mutp53 prostate cancer (PCa). A. Graphical representation comparing tumor sizes between mutant p53 (mutp53) and wild-type p53 (WTp53) mouse models (n = 7 per group). B. Time-course curves of tumor volume measurements for both the WTp53 and mutp53 groups (t test, p < 0.05). C. Comparative analysis of tumor weights between the mutp53 and WTp53 groups (t test, p < 0.001). D. Overview of genes differentially expressed in mutp53 versus WTp53 tumors. E. Hallmark pathway enrichment analysis of the differentially expressed genes revealed key signaling pathways altered in mutp53 tumors compared with WTp53 tumors. F-H. Immunohistochemical staining (F) and quantification of p-JAK2 (G) and p-STAT3 (H) expression in tumor tissues between the p53-mutated and p53-nonmutated groups. I. Western blotting assays showing increased levels of phosphorylated JAK2 and STAT3 in mutp53 PCa cells following IL-6 stimulation. Note: To compare two groups, a t test was used. The values are mean ± SD of three independent experiments.

Fig. 2. PHI inhibited the JAK/STAT and Notch1 signaling pathways in RAW264.7 cells. (a) The 3D and 2D models of molecular docking of PHI and JAK1. (b) The 3D and 2D models of molecular docking of PHI and JAK2. (c) The 3D and 2D models of molecular docking of PHI and Notch1. (d-g) The expression of JAK1, JAK2, STAT1 and Notch1 mRNA in RAW264.7 cells after treatment with LPS/IFNγ and PHI for 12 h was detected by RT-qPCR (n = 3). (h) The expression of JAK1, JAK2, p-JAK1, p-JAK2, STAT1, p-STAT1 and Notch1 proteins in RAW264.7 cells after treatment with LPS/IFNγ and PHI for 12 h was detected by western blotting. (i-l) The relative quantification of p-JAK1/JAK1, p-JAK2/JAK2, p-STAT1/STAT1, and Notch1 protein expression in western blotting results was analyzed by ImageJ software (n = 3). Results are presented as mean ± SD. ###P 

Fig. 2. NP16 down-regulated the expression of COX-2, p-JAK2 and p-STAT3, and inhibited the nuclear translocation of STAT3 in both U251 and U87MG cells. (A, B) western blot analysis of the expression of JAK2, p-JAK2, p-STAT3 and STAT3: the cells were exposed to NP16 at concentrations of 0.25, 1 and 4 mM for 8 h; western blot analysis of the expression of COX-2: the cells were exposed to NP16 at concentrations of 1, 2 and 4 mM for 24 h (C, D, E) Relative expression of the target protein in the cell lysate was calculated using the ImageJ image analysis software. Each error bar represents the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the DMSO group. (F, G) representative fluorescence microscopy images: the cells were exposed to NP16 at concentrations of 4 mM for 8 h. Compound NP16 inhibited nuclear translocation of STAT3 in both U251 and U87MG cells. (Magnification  60, scale bar ¼ 20 mm).