Kinesin-1 (KIF5B/KLC1), Slp1, Slp2a, and Slp3 expression in CTLs. (A) Relative quantification of KIF5A, KIF5B, KIF5C, KLC1, KLC2, and KLC3 transcripts in a real-time PCR with CTL mRNA. Transcript levels for each sample were expressed as a proportion of the mean value for KLC1. The data are representative of 2 independent experiments performed in duplicate. (B) A schematic representation of the Slp3 domains used to localize the peptide identified by mass spectrometry. The peptide sequence is shown. (C) Flag-Rab27a and GFP-Slp3 were coexpressed into 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and then separated by SDS-PAGE. Coprecipitated Rab27a and Slp3 were immunoblotted with anti-Flag and anti-GFP antibodies. The blots represent 3 independent experiments. (D) Top panel: confocal microscopy of CTLs transfected with GFP-Slp3. Cells were then fixed, permeabilized, and stained with anti-tubulin (in red). Bottom panel: confocal microscopy of CTLs transfected with GFP-Slp3 and DsRed-Rab27a. The lower insets represent an enlarged image. Scale bars represent 5 μm. All images of single cells were representative of > 60 cells observed over at least 4 independent experiments performed in duplicate. (E) Relative quantification of Rab27a, Slp1, Slp2, and Slp3 transcripts was performed by real-time PCR of CTL mRNA. Transcript levels for each sample were expressed as a proportion of the mean value of Rab27a. The data are representative of 2 independent experiments performed in duplicate.