Af568 conjugated phalloidin

Trypsinized CHO cells in Dulbecco’s PBS supplemented with Ca²⁺ and Mg²⁺, 1 g/l glucose, and 4 mM sodium bicarbonate were preincubated with inhibitors or vehicle for 10 min at 37°C as indicated prior to being plated for 30 min onto glass coverslips that had been coated with 150 μg/ml fibrinogen and blocked with 2% fatty acid–free BSA. Fixed, washed cells were stained with AF568-conjugated phalloidin (Thermo Fisher Scientific); random images were acquired at ×20 magnification (EVOS imaging system). Prior to measuring cell areas with ImageJ, binary images were thresholded, and the watershed feature was applied to define single cells.

Anti-HSP90 (clone 3H3C27), anti-SHIP1 (clone PICI-A5), FITC-conjugated rat anti-mouse GR1 (clone RB6-8C5), PE-conjugated rat anti-mouse/human CD11b (clone M1/70), PE/Cy7-conjugated rat anti mouse/human CD45 (clone 30-F11), PerCP/Cy5.5-conjugated rat anti F4/80 (clone BM8), APC-conjugated rat anti-B220 (clone RA3-6B2), PE/Cy7-conjugated rat anti CD3 (clone 17A2) and pacific blue-conjugated rat anti-LY6G (clone 1A8) were from BioLegend (London, UK); anti-PTEN (clone D4.3), anti-PKB (clone 11E7), anti-PKB T308 (clone C25E6) and anti-PKB S473 (clone D9E) were from Cell Signaling Technology (London, UK). Rabbit IgG (I8140) was obtained from Sigma. Anti-SHIP2 (AF5389) and PE-conjugated rat anti-CD64 (clone FAB20741P) were from R&D Systems (Abingdon, UK) and biotinylated anti-PI(3,4)P2 (z-B034) was from Echelon Biosciences (Salt Lake City, UT, USA); streptavidin-AF647, AF488-conjugated phalloidin, AF568-conjugated phalloidin, and secondary antibodies anti-rat AF488, anti-rabbit AF568 and anti-rabbit AF488 were obtained from Thermo Fisher Scientific (Loughborough, UK).