Fig. 4: FAK regulates inflammatory responses in macrophages through interacting with TAK1.

A RAW 264.7 (RAW) cells were pretreated with 1 μM PND-1186 for 1 h and then stimulated with 0.5 μg/mL LPS for 30 min. Cell lysates were analyzed for p-TAK1 (Ser412) and p-IKKα/β (Ser176/180) levels. Total TAK1 and IKKβ were used as control. B RAW cells were treated with 0.5 μg/mL LPS for 10 min. Complexes of FAK-TAK1 were detected by immunoprecipitation. C RAW cells were treated with 0.5 μg/mL LPS for the indicated times. Complexes of FAK-TAK1 were detected by immunoprecipitation. D RAW cells were pretreated with 1 μM PND-1186 for 1 h and then exposed to 0.5 μg/mL LPS for 10 min. Complexes of FAK-TAK1 were detected by immunoprecipitation. E RAW cells were pretreated with 2 μM Takinib for 1 h and then exposed to 0.5 μg/mL LPS for 30 min. The phosphorylated ERK, p38 and JNK were examined by western blot assay. Total ERK, p38, and JNK were used as control (TAKi = Takinib). F RAW cells were pretreated with 2 μM Takinib for 1 h and then stimulated with 0.5 μg/mL LPS for 30 min. Cell lysates were analyzed for p-IKKα/β and IκBα levels. Total IKKβ and GAPDH were used as control. G RAW cells were pretreated with 2 μM Takinib for 1 h and then exposed to 0.5 μg/mL LPS for 24 h. IL-6 proteins in the culture medium were measured by ELISA. Data normalized to total proteins and presented as % LPS [Mean ± SEM, 3 independent experiments; ***P < 0.001 compared to LPS]. H RAW cells were pretreated with 2 μM Takinib for 1 h and then exposed to 0.5 μg/mL LPS for 8 h. mRNA levels of IL-6 were measured. Data normalized to β-actin and expressed as % Ctrl [Mean ± SEM, 3 independent experiments; *P < 0.05 compared to LPS]. I RAW cells were transfected with FAK-expressing plasmid. After 24 h, levels of p-FAK and p-TAK1 were detected. Total FAK, TAK1, and GAPDH were used as control. Control cells were transfected with negative control/empty vector (NC = negative control, O/E = overexpression). J 3T3 cells were transfected with FAK-WT-Flag/FAK-Y397F-Flag/TAK1-Myc expressing plasmid, respectively. After 24 h, levels of p-TAK1 were detected using western blot, with total FAK, Flag, Myc, and GAPDH as controls. K, L Cell-free kinase assay showing rhFAK phosphorylates rhTAK1. rhTAK1 was incubated with rhFAK in the presence or absence of ATP (100 μM). The samples were separated by SDS-PAGE and western blotting was used to detect p-TAK1, TAK1, and FAK in panel K. Densitometric quantification of p-TAK1 levels was determined in panel L [Mean ± SEM, 3 independent experiments; **P < 0.01 compared to rhTAK1]. M, N FAK-expressing RAW cells were treated with 2 μM Takinib for 12 h. IL-6 (L) and TNF-α (M) proteins in the culture medium were measured by ELISA. Data normalized to total proteins and presented as fold difference compare to NC [TAKi = Takinib; Mean ± SEM, 3 independent experiments; **P < 0.01 and ***P < 0.001 compared to NC; ^#P < 0.05 and ^##P < 0.01 compared to O/E].