Plasma membrane targeting of SH4 fusion proteins depends on N-terminal acylation and can be measured using an image analysis tool. (A) Stable HeLa cells expressing GFP fusion proteins containing either wild-type or mutant forms of the SH4 domains of either HASPB or YES1 were induced for reporter protein expression and subjected to live cell confocal microscopy to determine the subcellular localization of the SH4 fusion proteins. (B) Stable HeLa cells expressing both SH4-HASPB-GFP and SH4-YES1-mCherry were treated with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Subcellular localization of SH4 fusion proteins was analyzed by live-cell confocal microscopy. (C) Wide-field images from stable HeLa cells expressing wild-type or mutant SH4 domains of HASPB (a) or from HeLa cells expressing SH4-HASPB-GFP under treatment with 2-BP (b) were analyzed with the automated image analysis tool and evaluated for the detection of hit phenotypes.
