primary rabbit anti-pstat3 (tyr705) (d3a7) xp  (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

Journal: Biomolecules

Article Title: Uncovering a Novel Role of ROR1 in the Epigenetic Regulation of Tumor Suppressor Gene CREB3L1 in Triple-Negative Breast Cancer Cells.

doi: 10.3390/biom15050734

Figure Lengend Snippet: Figure 5. ROR1 activated STAT3 phosphorylation in TNBC cells. (a) Western blot analysis comparing the protein expression of STAT3 and pSTAT3 in MDA-MB-231 and HCC 1806 cells with high ROR1 expression (scRNA-control) compared to the same cell lines with ROR1 knocked down (siROR1). (b) Quantification of Western blot analysis shows that ROR1 knockdown decreases phosphorylated STAT3 (* p < 0.05, n = 3). Statistical significance was determined through paired Student’s t-tests. (c) Hypothetical figure illustrating the potential signaling pathway that ROR1 is activating to silence CREB3L1 expression in TNBC.

Article Snippet: The primary antibodies were as follows: ROR1 (Cell Signaling, 16540, 1:1000, Cambridge, UK), CREB3L1 (Protein Tech, 11235-AP, 1:2000, Rosemont, IL, USA), DNMT3A (Cell Signaling, 32578, 1:1000, Cambridge, UK), DNMT3B (Cell Signaling, 57868, 1:1000, Cambridge, UK), DNMT1 (Cell Signaling, 5032, 1:1000, Cambridge, UK), STAT3 (Cell Signaling, 12640, 1:1000, Cambridge, UK), pSTAT3 (Cell Signaling, 9145, 1:1000, Cambridge, UK), and β-actin (Santa Cruz, sc-47778, 1:1000, Dallas, TX, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Knockdown

Journal: Biomolecules

Article Title: Uncovering a Novel Role of ROR1 in the Epigenetic Regulation of Tumor Suppressor Gene CREB3L1 in Triple-Negative Breast Cancer Cells.

doi: 10.3390/biom15050734

Figure Lengend Snippet: Figure 6. STAT3 phosphorylation had an inverse effect on CREB3L1 protein expression in TNBC. (a) Western blot analysis comparing the expression of STAT3, pSTAT3, and CREB3L1 in TNBC cells treated with scRNA to the same cell lines with ROR1 expression knocked down (siROR1). (b) Quantification of Western blot analysis (n = 3). Statistical significance was determined through paired Student’s t-test (* p < 0.05, ** p < 0.01). (c) Western blot analysis comparing the protein expression of ROR1, STAT3, pSTAT3, DNMT3A, DNMT3B, and CREB3L1 in naive MCF10A cells compared to the same cell line that has been transfected with IL-6 recombinant protein (* p < 0.05, ** p < 0.01, n = 3).

Article Snippet: The primary antibodies were as follows: ROR1 (Cell Signaling, 16540, 1:1000, Cambridge, UK), CREB3L1 (Protein Tech, 11235-AP, 1:2000, Rosemont, IL, USA), DNMT3A (Cell Signaling, 32578, 1:1000, Cambridge, UK), DNMT3B (Cell Signaling, 57868, 1:1000, Cambridge, UK), DNMT1 (Cell Signaling, 5032, 1:1000, Cambridge, UK), STAT3 (Cell Signaling, 12640, 1:1000, Cambridge, UK), pSTAT3 (Cell Signaling, 9145, 1:1000, Cambridge, UK), and β-actin (Santa Cruz, sc-47778, 1:1000, Dallas, TX, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Transfection, Recombinant