The SCAR complex is recruited to the edge of PIP3 patches. AX2 cells were transfected with a vector that expresses the PIP3 marker PH-CRAC-RFP and the SCAR–WAVE complex marker HSPC300-GFP. Cells were cultured axenically in HL5 medium and incubated for 3 h in SorMC buffer before imaging to reduce autofluorescence. Cells were transferred to an acid-washed glass-bottom dish (MatTek Corporation) and allowed to migrate randomly. Images were acquired sequentially on a spinning disk microscope in the following order: RFP, GFP, trans. Exposure time per channel was 90 ms, and one set of images was collected per second. The RFP channel was corrected for bleaching using standard methods.
