pstat3  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: RNase 7 and Th cytokines synergistically increase the secretion of interleukin-6 from keratinocytes

doi: 10.1038/s41598-025-04403-8

Figure Lengend Snippet: IL-24 secretion correlates with and depends on IL-6 secretion and signaling and IL-6 secreted from R7-stimulated cells activates STAT3 signaling. ( a ) We calculated the ratio of cytokine release from unstimulated and R7-stimulated HPK and plotted the IL-24 ratio against the IL-6 ratio. Statistics: Pearson correlation, significant with p = 0.004, n = 15. ( b ) HPK were starved for 4 h and then stimulated for 15 min with either 10 ng/mL rIL-6, 10 ng/mL Hyper-IL-6 (Hy-IL-6) or left unstimulated. Phosphorylation of STAT3 and total STAT3 protein levels were visualized by immunoblot in comparison to housekeeping GAPDH protein. ( c ) pSTAT3/STAT3 ratios of four HPK donors were quantified and statistical significance was analysed by paired t test; **p < 0.01. Bars indicate median. ( d , e ) HPK of 9 donors were seeded on a 24-well plate and were not stimulated (ctr) or stimulated with 5 µg/ml R7 either in the absence or in the presence of 2 µg/ml of a function-blocking antibody against IL-6. After 72 h, we measured IL-24 transcript levels relative to GAPDH transcripts (d) and IL-24 release by ELISA ( e ). Data in ( d , e ) was statistically analyzed by Friedman’s test followed by Dunn’s multiple comparisons test. *p < 0.05, **p < 0.01, ns, not significant. Bars indicate median. Relative IL6R ( f ) and IL6ST ( g ) expression of HPK (n = 6) after 48 h without (ctr) or with 5 µg/ml R7 was determined by qRT-PCR and compared by Wilcoxon matched-pairs signed rank ( f ) or paired t test ( g ). Bars indicate median. ( h ) HPK were starved for 4 h and then stimulated for 15 min either directly with 5 µg/ml R7 or 10 ng/ml rIL-6 or with spnts of HPK that had been stimulated for 48 h with 5 µg/ml R7 or left unstimulated, followed by either no treatment or an incubation with 2 µg/ml of a function-blocking IL-6 antibody or an isotype control antibody for 1 h at 37°C. Phosphorylation of STAT3 was analysed by immuno-blotting ( i ) Quantification of pSTAT3 relative to STAT3 bands (n = 5). Statistical analysis was performed with Friedman’s test followed by Dunn’s multiple comparisons test. *p < 0.05. Bars represent median.

Article Snippet: Proteins were probed using monoclonal rabbit anti-pSTAT3 antibody (pY705, D3 A7; cat. #9145), monoclonal mouse anti-STAT3 antibody (124H6; cat. #9139) or monoclonal mouse anti-GAPDH antibody (cat. #14 C10; Cell Signaling Technology, MA, USA) at 1:1000 dilution and labeled with secondary anti-rabbit IgG HRP-linked antibody (cat. #7074; Cell Signaling Technology, MA, USA) or anti-mouse IgG HRP-linked antibody (cat. #7076; Cell Signaling Technology, MA, USA) at 1:2000 dilution, respectively.

Techniques: Phospho-proteomics, Western Blot, Comparison, Blocking Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Incubation, Control