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Three hours postinfection, cells were lysed in 25?mM Tris-HCl (pH 7.5), 1?mM EDTA, 10% glycerol, 500?mM NaCl, 1% Triton-X, 1?mM Na3VO4, and protease inhibitors (lysis buffer). The infected cell lysate was incubated with GFP-trap beads (Chromotek) for 1?hr at 4��C. The beads were subsequently washed three times with lysis buffer containing first 500 and then 50?mM NaCl. Total input and immunoprecipitated samples were subjected to SDS-PAGE and immunoblot analysis. Human A549 cells infected with recombinant virus expressing FLAG-F11 for 3?hr were lysed in PBS containing 0.5% Triton-X and protease inhibitors. The cell lysate was incubated with INSRR anti-FLAG M2 Affinity resin (Sigma-Aldrich) for 1?hr at 4��C, and the beads were subsequently washed three times with PBS-lysis buffer. BMS-754807 research buy Total input and immunoprecipitated samples were subjected to SDS-PAGE and immunoblot analysis. To investigate the F11:Myosin-9A:RhoA complex, human A549 cells infected with recombinant virus expressing FLAG-F11 for 3?hr were lysed in Mg2+ lysis/wash buffer (Millipore) containing protease inhibitors. The cell lysate was incubated with anti-FLAG M2 Affinity resin (Sigma-Aldrich) for 1?hr at 4��C, and the beads were subsequently washed three times with Mg2+ lysis/wash buffer. Total input and immunoprecipitated samples were subjected to SDS-PAGE and immunoblot analysis. The following RNAi were used: All-Star control (QIAGEN), siGENOME Human Myosin9A siRNA (MYO9A-1, 5��-GAAAGAAGCUUAGCCCUUA-3��; and MYO9A-3, 5��-GAACAUACAUUACGGAUAU-3��) (Dharmacon), siRNA against the 3��UTR of human Myosin-9A (5��-CCUCUAUUUUAAGAUUUCU-3��). HeLa cells were transfected with 20?nM of each oligo using the HiPerfect fast-forward protocol (QIAGEN). After 48?hr, the cells were infected with WR or ��F11 www.selleckchem.com/products/blz945.html viruses. In addition, HeLa cells treated with Myosin-9A 3��UTR or control RNAi for 48?hr were also transfected with GFP-tagged Myosin-9A, R2098M, and ��PBM pEL expression vectors 1?hr postinfection. In both cases, infected cells were fixed 8?hr postinfection and processed for immunofluorescence to assess actin tail number as described previously (Arakawa et?al., 2007a). Data are presented as mean?�� standard error of the mean and were analyzed by ANOVA or Student��s t test using Prism 6.0 (GraphPad Software, CA). A p value of
