Fig 1: Enhanced CSK:Cav-1 interaction leads to increased CSK activity in Spry2-/- CD4+ T cells.(A) Immunoblot of basal Cbp/PAG-1 and Cav-1 levels in CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). Numerical values indicate relative densitometric quantification of Cbp/PAG-1 and Cav-1 normalized to GAPDH. (B) Immunoblots of Cav-1 from cytosol and membrane fractions of stimulated CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). Na+ K+ ATPase and Tubulin serve as loading controls for membrane and cytosolic fractions, respectively. (C) Proximity ligation assay of CSK and Cav-1 (red dots) in US and anti-CD3/CD28 stimulated (10 min) CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). Scale bar, 5 µm. (D, E) Relative abundance of spots depicted as frequency of cells with spots (% cells with spots) (D) and relative frequency of cells with designated number of spots (E). (F) Real-time PCR analysis of basal Cav-1 mRNA expression in splenic CD4+ T cells from Spry2+/+ and Spry2-/- mice (n = 3). (G) Immunoblots of Cav-1, pan-Ub, and actin from CD4+ T cells of Spry2+/+ and Spry2-/- mice cultured for 14 h in MG132 (5 µM) or chloroquine (50 µM) (n = 3). (H) p-CSK, CSK, p-ERK1/2, and ERK1/2 immunoblots of CD4+ T cells from Spry2-/- mice (n = 3) treated with a membrane-permeable AP-ScrP or AP-CSD. (I, J) CD4+ T cells from Spry2-/- mice (n = 3) treated with a membrane-permeable AP-ScrP or AP-CSD and cultured in CD3/CD28-coated plates for 48 h and analyzed for the release of IL2 and IFN?. Significance * p < 0.05, ** p < 0.005, by Student t test. All the data of this figure can be found in the S1 and S2 Data files. AP-CSD, antennapedia caveolin scaffolding domain; AP-ScrP, antennapedia scrambled peptide; Cav-1, caveolin-1; IFN-?, interferon gamma; IL2, interleukin 2; pan-Ub, pan-ubiquitin; Spry2, Sprouty2; US, unstimulated.
Fig 2: Enhanced interaction between CSK and LCK in Spry2−/− CD4+ T cells.(A, B) Representative confocal images showing localization of CSK (A) and enumeration of membrane localized CSK (B) in CD4+ T cells from Spry2+/+ and Spry2−/− mice (n = 3). (C) Immunoblot analysis of total CSK levels in Spry2+/+ and Spry2−/− CD4+ T cells (n = 2). (D) p-CSK and CSK immunoblots of cytosol and membrane fractions of stimulated CD4+ T cells from Spry2+/+ and Spry2−/− mice. Na+ K+ ATPase and Tubulin serve as loading controls for membrane and cytosolic fractions, respectively (n = 3). (E) Immunoprecipitation of LCK and immunoblotting of LCK, CSK, and p-CSK in CD4+ T cells from Spry2+/+ and Spry2−/− mice under unstimulated and anti-CD3/CD28-stimulated (10 min) conditions (n = 3). (F) Representative confocal images of LCK- and CSK-stained CD4+ T cells from Spry2+/+ and Spry2−/− mice under US and anti-CD3/CD28-stimulated (10 min) conditions. (n = 3) Scale bar, 5 μm. (G) A graphical presentation of Pearson correlation coefficient of colocalization of CSK with LCK, n = 25 cells per experimental group. (H) Representative confocal images of CD4 and p-LCK (Y505) immunofluorescence staining of lung sections from Asp-challenged Spry2+/+ and Spry2−/− mice from the asthma model. Scale bar, 10 μm (I) Statistical analysis of the CTCF intensity [represented as AU of p-LCK(Y505)] from individual CD4+ T cells shown in (H). Significance * p < 0.05, ** p < 0.005, by Student t test. (n = 3). All the data of this figure can be found in the S1 and S2 Data files. Asp, Aspergillus; AU, arbitrary unit; CTCF, corrected total cellular fluorescence; IB, immunoblotting; IP, immunoprecipitation; Spry2, Sprouty2; Sti, stimulated; US, unstimulated; WCL,whole cell lysate.
Supplier Page from Sino Biological, Inc. for Mouse CSK / C-Src kinase Protein (His & GST Tag)