Figure 6.

Rai14 interacts with myosin II and affects migration of BMDCs. (A) BMDCs transfected with either siRNA control or siRai14 were loaded in 5×8 μm micro-fabricated channels and imaged for 20 h in an epifluorescence Olympus microscope, using a 10X objective and acquiring one transmission phase image every minute. Representative kymographs are shown. (B) Quantification of the mean ± s.d cell speed (µm/min) from four independent experiments (n > 150 cells per condition). **P<0.01 (two-tailed paired Student’s t-test). (C) Quantification of mean ± s.d. speed fluctuations [calculated as s.d./mean instantaneous speed (7, 8, 23) from four independent experiments (n > 150 cells per condition). *P<0.01 (two-tailed paired Student’s t-test). (D) BMDCs were fixed and stained with antibodies against Rai14 (green) and Ii (red). Nuclei were stained with DAPI (blue). The image contrast and brightness have been increased to allow for easier visualization of the staining. The insets show magnifications of the boxed regions. Scale bars: 10 μm; inset: 2 μm. (E) Normalized fluorescent intensity profile for Rai14 and Ii along the lines as illustrated in the insets in D). Data represent the mean ± s.d. from two independent experiments. n = 78 vesicles from 37 cells. (F) DC lysates were subjected to immunoprecipitation with an antibody against Rai14 or an isotype control (IgG). Total lysate and immunoprecipitates (IP) were subjected to western blot analysis using antibodies specific to myosin II and Rai14. (G) Left panel: Coomassie Blue staining of bacterially expressed GST and GST–myosin II heavy chain tail purified using glutathione resin. Right panel: purified GST or GST–myosin II heavy chain tail were incubated with lysates from MelJuSo cells transfected with siRNA control or siRai14. Samples were subjected to affinity chromatography followed by western blot analysis using antibodies specific to GST, Rai14, and Ii.