FIGURE 2.

Effect of PI4,5P₂ and PIP₃ manipulation on the subcellular localization of C-terminal EGFP-tagged Lck-SH2. A, PI4,5P₂ and PIP₃ depletion greatly reduced PM localization of Lck-SH2. B, PI4,5P₂ depletion greatly inhibited PM localization of PI4,5P₂-selective EGFP-PLCδ-PH. C, K182A/R184A showed dramatically reduced PM localization, which was not affected by PI4,5P₂ depletion. D, R154A behaved similarly to WT. The PI4,5P₂ depletion was triggered by 2.5 μm A/C heterodimerizer that recruits FKBP-Inp to PM-anchored Lyn-FRB. All images for PI4,5P₂ depletion were taken before and 1 min after heterodimerizer treatment. PIP₃ depletion was achieved by pretreating the cells with 50 μm LY294002 for 1 h. HeLa cells were used for all measurements. Scale bars, 10 μm. Expression levels of EGFP-Lck-SH2 and EGFP-PLCδ-PH were kept as low as possible to minimize the possibility of non-physiological PM localization of proteins due to overexpression.