Pf 562271

Scratch wound migration assays were conducted using stably transfected MCF-7 cells. Confluent monolayers of transfected MCF-7 cells in 24-well plates were serum starved for 24 h to induce quiescence, and were then treated for 1 h with either serum free media, vehicle control, 70 nM UO126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; Abcam, Melbourne, VIC, Australia), or 1.5 nM PF-562271 (N-Methyl-N-[3-[[[2-[2-oxo-1,3-dihydroindol-5-yl)amimo]-5-(trifluoromethyl)pyrimidin-4-yl]amino)pyridine-2-yl)methanesulfonamide; Abcam). Following this, a scratch wound migration assay was performed as previously described (30 (link)).

Agrin (D2) and rabbit (H-300), Lrp4, Cdc42, N-WASP (western blot (WB) dilution—1:100) and anti-GFP (WB dilution—1:1,000) antibodies were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA), Antibodies against Na+/K+ ATPase, Rab5, integrin β1, cleaved caspase-3, total FAK, cortactin, pSrc, total Src, pPI3-K, total PI3-K, pAkt, total Akt, pERK1/2 and total ERK1/2 were obtained from Cell Signaling Technology (dilution for WB—1:1,000). Antibodies against pFAK pY397, caveolin-1, Ki67, E-cadherin, N-cadherin and Vimentin were purchased from BD Biosciences, San Jose, CA (WB dilution—1:500). PF-562271, glypican-3, flotillin-1, Arp2/3 subunit 1B, pFAK (pY397), Snail-1 and MuSK antibodies were obtained from Abcam (dilution for WB—1:500). Phospho-tyrosine monoclonal antibody 4G10, Agrin monoclonal antibody MAb5204, p34Arc, GFP and EGFP antibodies were obtained from Millipore, Billerica, MA (WB dilutions—1:1,000). Agrin polyclonal rabbit antibody, OKT-9 hybridoma cells producing anti-CD-71 antibody and fibronectin antibody were obtained from Sigma, St Louis, MO. Recombinant rat Agrin protein residues was obtained from R&D systems and My Biosource, Inc, San Diego, CA. Recombinant CTxB, Alexa 488 and 555 conjugated secondary antibodies were from purchased Molecular Probes, Invitrogen, Carlsbad, CA.

The human LUAC cell lines A549 and H1299 were purchased from American Type Culture Collection (ATCC) and maintained in RPMI‐1640 (KeyGen, Nanjing, China), except for SPCA‐1, which was maintained in DMEM (KeyGen, Nanjing, China), and both media were supplemented with 10% FBS (Life Technologies) at 37°C in a humidified atmosphere with 5% CO2. The FAK inhibitor PF-562271 (ab141360) was purchased from Abcam (Cambridge, UK). Cells were authenticated by STR analysis at Guangzhou Cellcook Biotech Co., Ltd. (Guangzhou, China) Characterized Cell Line Core Facility within the last three years and routinely tested negative for mycoplasma contamination.