src family kinase specific mab  (Cell Signaling Technology Inc)

Journal: International archives of allergy and immunology

Article Title: Phospho-proteomic analysis of interleukin (IL)-13 signaling in airway cells reveals SRC family kinase involvement in IL-13–induced inflammatory responses

doi: 10.1159/000549040

Figure Lengend Snippet: (a,b) Primary lung fibroblasts isolated from C57BL/6 mice were serum-starved for 4 hours, pretreated with 200 nM Dasatinib for 1 hour, and then stimulated with IL-13 (10 ng/mL) for the indicated time points. Cells treated with PDGF (20 ng/mL, 15 minutes) served as a positive control for SRC activation. Whole-cell lysates were analyzed by Western blot for pSRC, total SRC, pSTAT6, total STAT6, and β-actin. Densitometric analysis of pSTAT6/STAT6 is shown. n = 6–10. Statistical significance was determined by two-tailed Student’s t-test comparing each time point to baseline (0 min); *p < 0.05. (c,d) Primary human lung fibroblasts were treated under similar conditions and analyzed for pSRC, total SRC, pSTAT6, total STAT6, and GAPDH. n = 2. Levels of total SRC and total STAT6 were assessed simultaneously on the same Western blot, while levels of pSRC and pSTAT6 were measured simultaneously on different blots. The same amount of protein, from the same lysate were used to load blots of total, and phospho-proteins. Western blots for total proteins were stripped and re-probed to assess GAPDH/β-actin expression.

Article Snippet: Phospho-SRC (pSRC at Tyr416) was measured using an SRC family kinase-specific mAb (Cell Signaling Technology, #2101S).

Techniques: Activation Assay, Isolation, Positive Control, Western Blot, Two Tailed Test, Expressing

Journal: International archives of allergy and immunology

Article Title: Phospho-proteomic analysis of interleukin (IL)-13 signaling in airway cells reveals SRC family kinase involvement in IL-13–induced inflammatory responses

doi: 10.1159/000549040

Figure Lengend Snippet: (a,b) Primary mouse lung fibroblasts were serum-starved for 4 hours, pretreated with or without 200 nM Dasatinib for 1 hour, and then stimulated with IL-13 (10 ng/mL) for 30 minutes. Whole-cell lysates were analyzed by Western blot using antibodies against pSRC (Y416), total SRC, pSTAT6, and total STAT6. Densitometric analysis of pSTAT6/STAT6 is shown. n = 4. (c–e) A549 human airway epithelial cells were serum-starved for 18 hours, pretreated with or without 200 nM Dasatinib for 1 hour, and then stimulated with IL-13 (10 ng/mL) for 20 hours. RT-qPCR was performed to assess expression of C3 , CCL24 , and ALOX15 . n = 10–12. (f–k) Primary mouse lung fibroblasts were serum-starved for 18 hours, pretreated with or without 10 nM SRC inhibitor I for 1 hour, and then stimulated with IL-13 (10 ng/mL) for 20 hours. RT-qPCR was performed to assess expression of C3 , Ccl24 , Fizz1 , Cxcl1 , Mmp9 , and Sprr2 . n = 5–6. Data are shown as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test; p < 0.05, *p < 0.01, **p < 0.001. Levels of pSTAT6, total SRC, and GAPDH were assessed on the same blot after stripping and reprobing. Levels of total STAT6 and pSRC were assessed on separate blots. The same amount of protein, from the same lysate were used to load all blots.

Article Snippet: Phospho-SRC (pSRC at Tyr416) was measured using an SRC family kinase-specific mAb (Cell Signaling Technology, #2101S).

Techniques: Inhibition, Phospho-proteomics, Gene Expression, Western Blot, Quantitative RT-PCR, Expressing, Stripping Membranes