Confocal microscopy of LAD-III B cells transfected with human KINDLIN3-GFP cDNA constructs. (A) FACS-sorted LAD-III B cells transfected with GFP cDNA were compared with cells transfected with WT KINDLIN3, Gly308Arg, Pro425ArgAlaX, and double mutant KINDLIN3-GFP cDNAs. Fluorescent images of live B cells taken at the interface with ICAM-1 show punctate membrane localization (arrows) of WT and Gly308Arg Kindlin-3-GFP protein compared with other transfectants showing a diffuse distribution of fluorescence in the cell body and extensions. The boundaries of the cells are highlighted with a white line. Only the WT and Gly308Arg KINDLIN3-GFP cDNA–transfected cells adhered well. The cells are typical examples from 1 experiment of n = 4. Scale bar represents 10 μm. (B) Western blot of GFP, WT kindlin-3-GFP, full-length Gly308Arg, and truncated Pro425ArgAlaX and double mutant kindlin-3-GFP proteins expressed in EBV-transformed B cells and showing the expected molecular size. Loading control is α-tubulin.