Association of MyH9 with LFA-1. (A) Human primary T lymphocytes were allowed to adhere to cover glasses coated with ICAM-1 (IC-1) or poly-l lysine (PLL) ± CXCL-12. Cells were suspended in either L-15/2 mg/ml glucose or 20 mM Hepes, 150 mM NaCl, 5 mM MgCl2, 1 mM EGTA, and 2 mg/ml glucose, as indicated. Migrating T lymphocytes were tracked over a 20-min period, and time-lapse DIC images were acquired every 5 s to generate movies (Videos S1–4). The bottom left corner of each image shows a randomly selected region at threefold magnification. Bar, 100 μm. (B and C) LFA-1 immunoprecipitates obtained from bound cells (using TS2/4 antibody) were analyzed for possible LFA-1 binding partners. MyH9 was identified by silver staining (n = 3) (B) and mass spectrometry (C). The spectrum of one of the MyH9 peptides obtained by nanospray-ion trap tandem mass spectrometry is shown in C. (D) PCR amplification of MyH9, MyH10, and MyH14 cDNAs from human PBMCs and T lymphocytes. Reverse-transcribed cDNA from human skeletal muscle served as a positive control. (E) LFA-1 immunoprecipitates obtained for each of the four substrate conditions were subjected to Western blotting with the indicated antibodies.
