Loss of PDGF-induced activation of tyrosine kinases in isolated primary cardiac fibroblasts from β3−/− mice. Sundaravadivel Balasubramanian Lakeya Quinones Harinath Kasiganesan Yuhua Zhang Dorea L. Pleasant Kamala P. Sundararaj Michael R. Zile Amy D. Bradshaw Dhandapani Kuppuswamy 10.1371/journal.pone.0045076.g005 https://plos.figshare.com/articles/figure/_Loss_of_PDGF_induced_activation_of_tyrosine_kinases_in_isolated_primary_cardiac_fibroblasts_from_946_3_8722_8722_mice_/249706 <p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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. <i>Scale bar, 10 µm</i>. (<b>E</b>) WT cardiac fibroblasts were plated on glass coverslips in DMEM containing 10% FBS. After overnight incubation, the cells were either uninfected or infected with <i>β-gal,</i> 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. <i>Scale bar, 20 µm.</i></p> 2013-02-19 23:34:13 pdgf-induced activation tyrosine kinases cardiac fibroblasts