Fig. 7

Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (siGRN) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in siRYK- or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK, their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with siRYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells (G), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (siGRN) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK-specific siRNA or control (siCTR) siRNA (I) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout