IRSp53 binds to negatively charged SLBs. Representative fluorescence images showing that IRSp53 WT binds to lipid membrane containing 2% PI(4,5)P2, whereas the Lys-to-Glu mutations in the BAR domain (IRSp53_K4E) abolished its lipid-binding ability. Mutation of positive charges in the SH3 domain (IRSp53_RKE) did not interfere with its lipid membrane binding. The starting concentration of IRSp53 WT or mutant proteins in solutions was at 2 μΜ. 10% of protein was labeled with Alexa Fluor 488 C₅ maleimide dye.