Eight well glass bottom μ slide

PI3KC2α KD Cos7 cells or PI3KC2α KD Cos7 cells re-expressing eGFP-PI3KC2α WT or mutants were grown on Matrigel (BD Biosciences)-coated eight-well glass bottom μ-slide (ibidi). Cos7 cells were washed with PBS containing 10 mM MgCl₂ once and fixed in 2% PFA with 0.5% glutaraldehyde for 20 min at room temperature. Cells were washed three times with PBS and twice with PBS containing 50 mM NH₄Cl. Cells were permeabilized with PBS containing 0.5% Saponin and 1% BSA for 30 min. PI(3,4)P2 antibody (Echlon catalog no. Z-P034b) and Goat anti-Mouse IgG (H + L) AF647 labeled secondary antibody (Thermo Fisher catalog no. A21237) were incubated for 2 and 1 h, respectively, in PBS buffer containing 1% BSA and 10% normal goat serum. Cells were analyzed by total internal reflection fluorescence microscopy (Nikon TI Eclipse, 488 and 561 nm laser, ×60 numerical aperture 1.49 objective and sCMOS Andor mNeo). Plasma membrane PI(3,4)P₂ levels at clathrin-coated pits were quantified using ImageJ software.