Naive CD8⁺ T cells possess an intracellular pool of LFA-1. (A) Schematic of CD11a-mYFP mouse generation. The mYFP sequence was knocked into the C terminus of the mouse integrin CD11a subunit. (B) CD11a PCR depicting the increased size of CD11a corresponding with the mYFP tag. (C) Corresponding size increase was also detected in a silver stain at the protein level. mAb M17/4 was used for immunoprecipitation of CD11a. (D) Western blot analysis of YFP expression in YFP immunoprecipitate (IP) and total cell lysate from splenocytes of CD11a-mYFP mice showing the intact YFP conjugation to CD11a. No evidence of proteolytic cleavage of YFP was detected. (E) Representative images of permeabilized naive CD8⁺ T cells stained with CD11a (LFA-1) or α-tubulin antibodies on noncoated glass surface (unstimulated/round), after 30 min of migration on ICAM-1 and CCL21 coating (migration) or after 60 min of conjugation with N4-pulsed BMDCs (conjugation) showing the intracellular pool of LFA-1. Bars, 2 µm. Graph shows colocalization of YFP signal versus anti–CD11a antibody (LFA-1) signal in naive CD11a-mYFP CD8⁺ T cells. Pearson coefficient was generated as YFP (CD11a-mYFP)/red (anti-CD11a). Note that the YFP signal and the anti–CD11a antibody (LFA-1) signal are highly colocalized in naive CD11a-mYFP CD8⁺ T cells. Data are presented as mean ± SEM; n = 4 mice/group (10–20 cells per mouse). Note that there are equivalent levels of total LFA-1 (intracellular staining and surface LFA-1) detected in both saponin and Triton X-100 permeabilized samples (Fig. S2 C).