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Figure 5: AM966-induced phosphorylation of VE-cadherin is not FAK-dependent. (a) HLMVECs (~70% confluent) transfected with siLPA1 or sicont were treated with AM966 (1.0 μM) or DMSO for 30 min. Cell lysates were immunoblotted with phosphospecific FAK (P-FAK), total FAK, and LPA1 antibodies. (b) Analysis of P-FAK by densitometry of the results in (a) was performed by Image J software (), and statistical analysis was shown. (c) Confluent HLMVECs were pretreated with FAK kinase inhibitor (0 to 10.0 μM) for 1 h, and then cells were treated with DMSO or AM966 (1.0 μM) for an additional 30 min. Cell lysates were immunoblotted with P-VE-cadherin and total VE-cadherin antibodies. (d) Analysis of P-VE-cadherin by densitometry of the results in (c) was performed by Image J software (), and statistical analysis was shown. Shown are representative blots from three independent experiments. (e) Confluent HLMVECs were plated on gold microelectrodes and pretreated with FAK inhibitor (1.0 or 5.0 μM) for 1 h and then stimulated by 1.0 μM AM966 or DMSO. The TEER tracing represents pooled data (±SEM) from 3 independent experiments. (f) The resistance in response to AM966 treatments during indicated time period (e) was quantified and statistical analysis was performed.