phospho src  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5

doi: 10.1016/j.jbc.2026.111190

Figure Lengend Snippet: Cav1 tyrosine (Y)-14 phosphorylation induced by APC–PAR1 is mediated by c-Src. A – C, endothelial EA.hy926 cells were pretreated with dasatinib or DMSO prior to addition of APC. Cell lysates were then immunoblotted to detect Cav1 Y14 and c-Src Y416 phosphorylation as indicated. D – G, endothelial cells transfected with nonspecific (NS) or c-Src-specific siRNA were treated with or without APC, lysed, and immunoblotted as indicated. β-tubulin and GAPDH were used as loading controls. The data were quantified (mean ± SD) from four independent biological replicates and expressed as the fraction relative to the untreated control and analyzed by two-way ANOVA followed by Šídák's multiple comparisons test. ( B ) ∗ p = 0.0136; ( C ) ∗∗ p = 0.0038; ( F ) ∗∗∗ p = 0.0003; and ( G ) ∗ p = 0.0125. Student's unpaired t test, ( E ) ∗∗∗ p = 0.0002. APC, activated protein C; Cav1, caveolin-1; DMSO, dimethyl sulfoxide; PAR1, protease-activated receptor 1.

Article Snippet: In this study, the following antibodies were used: mouse anti–PAR1 WEDE (Beckman Coulter, #IM2584), anti-Cav1 (CST, #3267S and BD, #610060), anti-Cav1 Y14 phospho antibody (CST, #3251), anti-βarr2 (Abcam, #ab54790), GAPDH (GeneTex, #GT239), c-Src Y416 (CST, #2101), anti-c-Src (CST, #2109), GRK5 (Santa Cruz, #sc-518005), GRK5 polyclonal antibody (Invitrogen, #PA5-96262) anti-GRK4-6 (Millipore, #05-466), anti-HA (CST, #3724S), anti-rabbit IgG (CST, #2729), anti-β-Tubulin (CST, #86298), anti–early endosome antigen-1 (BD Biosciences, #610457), anti-Vinculin (Sigma, #V9131), and anti-HA conjugated to horseradish peroxidase (Roche, #11667475001).

Techniques: Phospho-proteomics, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5

doi: 10.1016/j.jbc.2026.111190

Figure Lengend Snippet: βarr2 is required for APC-stimulated c-Src tyrosine (Y)-416 phosphorylation and Cav1 Y14 phosphorylation. Endothelial EA.hy926 cells were transfected with nonspecific (NS) or βarr2-specific siRNA, treated with APC, and c-Src Y416 phosphorylation ( A and B ) and Cav1 Y14 phosphorylation ( C and D ) were detected by immunoblotting as indicated. GAPDH was used as a loading control. The data were quantified (mean ± SD) from four independent biological replicates and expressed as the fraction relative to the untreated control and analyzed by two-way ANOVA followed by Šídák's multiple comparisons test. B, NS siRNA with and without APC, ∗∗ p = 0.0097; ns = not significant. D, NS siRNA with and without APC ∗∗∗ p = 0.0009; βarr2 siRNA with and without APC, ∗∗ p = 0.0061. APC, activated protein C; βarr2, β-arrestin-2; Cav1, caveolin-1.

Article Snippet: In this study, the following antibodies were used: mouse anti–PAR1 WEDE (Beckman Coulter, #IM2584), anti-Cav1 (CST, #3267S and BD, #610060), anti-Cav1 Y14 phospho antibody (CST, #3251), anti-βarr2 (Abcam, #ab54790), GAPDH (GeneTex, #GT239), c-Src Y416 (CST, #2101), anti-c-Src (CST, #2109), GRK5 (Santa Cruz, #sc-518005), GRK5 polyclonal antibody (Invitrogen, #PA5-96262) anti-GRK4-6 (Millipore, #05-466), anti-HA (CST, #3724S), anti-rabbit IgG (CST, #2729), anti-β-Tubulin (CST, #86298), anti–early endosome antigen-1 (BD Biosciences, #610457), anti-Vinculin (Sigma, #V9131), and anti-HA conjugated to horseradish peroxidase (Roche, #11667475001).

Techniques: Phospho-proteomics, Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5

doi: 10.1016/j.jbc.2026.111190

Figure Lengend Snippet: Model of PAR1–GRK5–βarr2–c-Src regulation by Cav1. A substantial population of PAR1–Cav1 and GRK5–Cav1 complexes coexist at the plasma membrane under basal conditions. APC bound to EPCR cleaves and activates PAR1, resulting in GRK5-dependent phosphorylation of the receptor C-terminal tail within 30 min. This may occur during a time when GRK5 dissociates from Cav1. Next, APC-activated and phosphorylated PAR1 recruits βarr2, which promotes activation of c-Src Y416 phosphorylation and c-Src-dependent Cav1 Y14 phosphorylation, a process that occurs after 30 min and is sustained through 90 min. At 30 min, GRK5–Cav1 may reassociate, whereas activated PAR1 and Y14 phosphorylated Cav1 remain dissociated through the 90 min interval. APC, activated protein C; βarr2, βarr2; Cav1, caveolin-1; EPCR, endothelial protein C receptor; GRK5, GPCR kinase 5; PAR1, protease-activated receptor-1.

Article Snippet: In this study, the following antibodies were used: mouse anti–PAR1 WEDE (Beckman Coulter, #IM2584), anti-Cav1 (CST, #3267S and BD, #610060), anti-Cav1 Y14 phospho antibody (CST, #3251), anti-βarr2 (Abcam, #ab54790), GAPDH (GeneTex, #GT239), c-Src Y416 (CST, #2101), anti-c-Src (CST, #2109), GRK5 (Santa Cruz, #sc-518005), GRK5 polyclonal antibody (Invitrogen, #PA5-96262) anti-GRK4-6 (Millipore, #05-466), anti-HA (CST, #3724S), anti-rabbit IgG (CST, #2729), anti-β-Tubulin (CST, #86298), anti–early endosome antigen-1 (BD Biosciences, #610457), anti-Vinculin (Sigma, #V9131), and anti-HA conjugated to horseradish peroxidase (Roche, #11667475001).

Techniques: Clinical Proteomics, Membrane, Phospho-proteomics, Activation Assay