Fig. 4.

Sustained FAK phosphorylation mediates spreading in Atgl−/− macrophages in a Src kinase-dependent manner. a Total RNA was isolated from Wt, Hsl−/− and Atgl−/− macrophages, reverse transcribed and mRNA expression of FAK was determined by real-time PCR including normalization to hypoxanthine guanine phosphoribosyl transferase (HPRT). Data represent mean values (n = 4) of two independent experiments ±SEM performed in triplicate repeats. ***p ≤ 0.001. b Western blot analysis in macrophage lysates from Wt, Hsl−/− and Atgl−/− mice using antibodies specific for total and phosphorylated (p)FAK (Tyr576/577). c, e Wt, Atgl−/− and (d) Wt macrophages loaded with VLDL (150 μg/ml for 18 h) were incubated with LPA (9 ng/ml) for 0, 2, 5 and 10 min. The lysates were subjected to Western blot analysis using antibodies specific for (c, d) total and pFAK and (e) total and pSrc (Tyr416). f Atgl−/− macrophages were preincubated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2, 10 μM) for 30 min and then exposed to SDF-1 (60 ng/ml) for 0, 2, 5 and 10 min. Western blot analysis was performed using the above-mentioned antibodies. g, h The lysates were subjected to Western blot analysis using antibodies specific for (g) total and pERK2 and (h) total and pSHP-2 (Tyr580). Ratios of two Western blots were calculated by dividing band intensities of phosphorylated to total protein expression. Fold changes were calculated relative to the ratios of unstimulated cells