Fig. 5. Expression of FRNK suppresses phosphorylation of FAK at Tyr³⁹⁷ and K1735 cell motility. M2 and M2GFP-FRNK cell lysates were immunoprecipitated with anti-FAK and then analyzed by western blotting with anti-pTyr³⁹⁷ antibodies (top). The membrane was stripped and reprobed with anti-FAK antibodies to ensure that amounts of protein were equivalent (middle). Detection of the FRNK construct was evaluated by a western blot of whole cell lysate with antibodies to the C-terminus of FAK (lane 2, bottom). (B) M2 and M2GFP-FRNK cells were seeded onto VN-coated filters and monitored for migration over a 2 hour period or plated onto RBM-coated filters and allowed to invade overnight, as described in Materials and Methods. The results (means±s.e.m.) are representative of three independent experiments each performed in triplicate. The average number of M2 cells migrating on VN was 238 (±11.5), whereas, the average number of migrating M2GFP-FRNK cells was 11.5 (±1). Similarly, invasion of an RBM was suppressed approximately 80% by expression of FRNK (B). The average number of M2 cells invading was 46 (±19), whereas only 8 (±1) M2GFP-FRNK cells invaded. These results suggest that phosphorylation of FAK on Tyr³⁹⁷ is required for maximum K1735 cell motility.