Loss of PDGF-induced activation of tyrosine kinases in isolated primary cardiac fibroblasts from β3−/− mice.
(A) Equal numbers of WT and β3−/− cardiac fibroblasts were allowed to adhere for 2 h in 35 mm culture dishes. Cells were stimulated with PDGF (10 ng/mL) for the indicated durations following overnight serum starvation. Triton-X-100 soluble cell extracts were analyzed by Western blotting using indicated phospho-specific antibodies. Individual total proteins are shown at the bottom. Blots are representative of data from three independent experiments. (B) The ratio of phosphorylated to total proteins in the case of PDGFR and Pyk2 are shown as average ± SEM. # p<0.05 vs. control; @ p<0.05 vs. WT 5 min. (C) Cell lysates from CFb as shown in (A) above were immunoblotted with indicated phospho-specific MAPK pathway members. Individual total proteins are shown at the bottom. Blots are representative of data from three independent experiments. (D) WT and β3−/− cardiac fibroblasts were serum starved for 16 h and then treated with PDGF (10 ng/mL) for indicated durations and fixed with 1% paraformaldehyde. The cells were then stained with phospho-PDGFR (α-Y849/β-Y857) (Red) and phosphor-Pyk2 (Y402) (Grey) antibodies and imaged under confocal microscopy using 60X oil objective. Scale bar, 10 µm. (E) WT cardiac fibroblasts were plated on glass coverslips in DMEM containing 10% FBS. After overnight incubation, the cells were either uninfected or infected with β-gal, Y402F Pyk2 (402 Pyk2) or β3-integrin (β3 Intg) at an MOI of 50. After 36 h of infection, the cells were fixed and stained for fibronectin (Green) and actin (Grey) and subjected to confocal microscopy as shown under (D). Representative images were from at least three independent experiments. Scale bar, 20 µm.