Biocoat poly l lysine coated coverslips

The HeLa clone with regulatable expression of HA-DOCK10 was grown onto 12 mm BioCoat Poly-L-Lysine-coated coverslips (Corning Inc., Corning, NY, USA). Preparations were labelled using the F-actin Visualization Biochem Kit (Cytoskeleton Inc., Denver, CO), following the manufacturer’s instructions with previously described modifications [9 (link)]. Briefly, after fixation and permeabilization, coverslips were incubated for 1 h at 4 °C with one of two primary Abs, either a rabbit anti-DOCK10.1 antiserum raised against peptide MAGERTRRFTRSLLRPGQAAEL [21 (link)] or a rat anti-HA Ab (Table 2). Following three washes, coverslips were simultaneously labelled with one of two secondary Abs, either anti-rabbit Igs-FITC Igs or anti-rat Igs-Alexa Fluor 488 (Table 2), 100 nM phalloidin-rhodamine (TRITC), and 1 µg/mL DAPI for 30 min at room temperature. After the final washing steps, coverslips were placed onto slides using Dako fluorescent mounting medium. Fluorescence images were acquired by an Eclipse Ti inverted microscope (Nikon Instruments Inc., Melville, NY, USA) using the NIS Elements software.