anti src family kinases  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src phosphorylates free TAB1 not bound to TAK1. A , Crystal structure of the TAK1-TAB1 fusion protein (PDB id: 2EVA ). An enlarged version of the interaction interface shows Y125 and R225 of the TAK1 kinase domain forming a hydrogen bond ( yellow dashed line ) with TAB1-Y481. The distance between the backbone amide nitrogen atom of Y125 and the side chain oxygen atom of Y481 is 3.4 Å, while the distance between the guanidinium nitrogen atom (Nη1) of R225 and the side chain oxygen atom of Y481 is 3.1 Å. The figure was generated using PyMol (DeLano Scientific; www.pymol.org ). B , COS-7 cells were transfected with EGFP-TAB1-C or TAK1-TAB1 with Src. C , COS-7 cells were transfected with EGFP-TAB1, Flag-TAK1, and Src. Cell lysates or anti-GFP immunoprecipitates were immunoblotted with primary antibodies against pY481-TAB1, EGFP, pT187-TAK1, TAK1, and pY419-Src. D , the relative quantification of pY481-TAB1 in lysates, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01.

Article Snippet: Primary antibodies for pY419-Src family kinase (2101S), total-Src (2110S), pT180/pY182-p38 (4511S), pY701-STAT1 (sc-136229), total-STAT1 (sc-346), pY576/577-FAK (3281S), and total-FAK (3285S) were purchased from Cell Signaling Technology. pS75-Src (ab79308) was from Abcam.

Techniques: Generated, Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 directly phosphorylates Src to increase its kinase activity. A , comparison between the substrate consensus sequence of p38α and the amino acid sequences of human Src around S75. Asterisks indicate amino acids that are common to both. B , an in vitro kinase assay using recombinant His-tagged Src and GST-tagged p38α proteins. Immunoblot analyses were performed with the primary antibodies indicated. C , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗∗ p < 0.01. D , COS-7 cells were transfected with Src and p38α (WT, CA or KD), and then treated with SB203580 for 5 h. Immunoblot analyses were performed with the primary antibodies indicated. E , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01. F , Src immunoprecipitated from transfected COS-7 cells was preincubated with recombinant p38 for 60 min. After removal of p38, the tyrosine kinase activity of Src toward GST-TAB1-C at Y481 was evaluated by an additional 20-min incubation. The relative quantification of pY481-TAB1, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗ p < 0.05. G , COS-7 cells were transfected with EGFP-TAB1, Src (WT or S75A), and p38α. Immunoblot analyses were performed with the primary antibodies indicated. H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of four independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05.

Article Snippet: Primary antibodies for pY419-Src family kinase (2101S), total-Src (2110S), pT180/pY182-p38 (4511S), pY701-STAT1 (sc-136229), total-STAT1 (sc-346), pY576/577-FAK (3281S), and total-FAK (3285S) were purchased from Cell Signaling Technology. pS75-Src (ab79308) was from Abcam.

Techniques: Activity Assay, Comparison, Sequencing, In Vitro, Kinase Assay, Recombinant, Western Blot, Quantitative Proteomics, Two Tailed Test, Transfection, Immunoprecipitation, Incubation

Journal: PLOS Genetics

Article Title: Fibroblast mechanoperception instructs pulmonary developmental and pattern specification gene expression programs

doi: 10.1371/journal.pgen.1011924

Figure Lengend Snippet: ( A ) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP following stimulation with 20μM SRC specific inhibitor KB SRC 4 or 10μM pan-SFK inhibitor PP2 for 6h. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to DMSO vehicle control. Mean ± S.D. plotted; N = 3 (Thy-1 KO -5kPa and WT-1GPa) or N = 4 (WT-5kPa and Thy-1 KO -1GPa) independent experiments; WT-5kPa and WT-1GPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. ( B ) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP substrates coated with αV-promoting (Fn 4G ) or α5-promoting (Fn 9*10 ) peptide fragments of the 9–10FnIII integrin binding site. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to full length human Fn (Fn Full ) control. Mean ± S.D. plotted; N = 3 (WT-5kPa, WT-1GPa, and Thy-1 KO -5kPa) or N = 4 (Thy-1 KO -1GPa) independent experiments; WT-5kPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa = Brown-Forsythe and Welch ANOVA with post-hoc unpaired t-tests with Welch’s correction, WT-1GPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. For all statistical tests: ns = p > 0.05; p < 0.05 (*); p < 0.01 (**); p < .001 (***); p < .0001 (****).

Article Snippet: After allowing for adhesion, cells were stimulated with 1% FBS DMEM containing 20μM SRC inhibitor KB SRC 4 (Tocris) or 10μM pan-Src Family Kinase (SFK) inhibitor PP2 (Tocris) for periods of 3h or 6h.

Techniques: Western Blot, Cell Culture, Control, Binding Assay

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