Alexa fluor 350 labeled phalloidin

Cells grown on glass coverslips and exposed to various agents were fixed with 3.7% paraformaldehyde and permeabilized with 0.25% Triton X-100. After blocking of nonspecific sites with 2.5% BSA in PBS, the cells were immunostained with antibodies to Myo1E (1/200 dilution) or to SH3P2 (1/200), and immune complexes were detected with Alexa Fluor 488– or 546–conjugated goat antibodies to rabbit IgG (1/200 dilution; Molecular Probes). F-actin was visualized with the use of Alexa Fluor 350–labeled phalloidin (Molecular Probes). Images were acquired with an Axiovert 200M microscope equipped with a FluoArc mercury lamp, a Plan-NEOFLUAR 40×/0.75 objective lens, and an AxioCam MRm camera (ZEISS). For comparison of the localization of Myo1E or of EGFP-Myo1E and its mutants, images were acquired with the same exposure time in respective experiments (typically 2 s for Myo1E and 5 s for EGFP). Images were analyzed with AxioVision software version 4.8.2 (ZEISS).

Anti–NMHC-IIB and anti–NMHC-IIA polyclonal antibodies targeting the carboxyl terminus of NMHC-IIB and NMHC-IIA, respectively, were used as previously described (Saitoh et al., 2001 ; Sato et al., 2007 (link)). An anti-vinculin monoclonal antibody (hVIN-1) and FITC-conjugated phalloidin were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 350–labeled phalloidin was purchased from Molecular Probes (Eugene, OR). Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) and Cy3-conjugated goat anti-mouse IgG (H+L) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Fibronectin purified from human plasma was purchased from Roche Diagnostics (Basel, Switzerland).