Figure 3. Chemotactic responses are... | Rockefeller University Press

Figure 3.

$Figure 3. Chemotactic responses are altered in cells with prolonged PKBR1 signaling. (A) ACA activation upon uniform cAMP stimulation was measured in rasC− cells or rasC− cells induced to express Flag-RasC or -RasCQ62L. The data represent the range (mean ± standard error) of two independent experiments. The basal activities ranged from 1.9 pmol/min/mg protein for rasC− to 2.5 pmol/min/mg protein for Flag-RasC/rasC− and 3.8 pmol/min/mg protein for Flag-RasCQ62L/rasC− cells. (B) Actin polymerization was measured in wild-type (WT) cells or rasC− cells expressing Flag-RasC or -RasCQ62L. Cells were stimulated with cAMP and lysed with Triton X-100 buffer. The Triton-insoluble pellet fraction was analyzed by SDS-PAGE and Coomassie blue staining. The amount of actin in the pellet was quantified by densitometry. The data represent mean ± SD of three independent experiments. A.U., arbitrary unit. (C) The translocation of LimEΔcoil-RFP was recorded by fluorescence microscopy with images taken every 3 s. Frames from the indicated time points after the addition of cAMP are shown. (D) The chemotactic movements to a micropipette releasing 1 µM cAMP (black dot) were recorded by time-lapse microscopy. Images from frames at 2.5-min intervals were processed to outline the cells, color coded for each time point, and overlaid. (E) The expression of cAR1 is comparable in rasC− cells induced to express Flag-RasC or -RasCQ62L. At the indicated time points after initiation of development, CHAPS-insoluble membrane fractions were prepared as described previously (Xiao et al., 1997) and probed with anti-cAR1 antibody. Bar, 10 µm.$

Chemotactic responses are altered in cells with prolonged PKBR1 signaling. (A) ACA activation upon uniform cAMP stimulation was measured in rasC⁻ cells or rasC⁻ cells induced to express Flag-RasC or -RasC^Q62L. The data represent the range (mean ± standard error) of two independent experiments. The basal activities ranged from 1.9 pmol/min/mg protein for rasC⁻ to 2.5 pmol/min/mg protein for Flag-RasC/rasC⁻ and 3.8 pmol/min/mg protein for Flag-RasC^Q62L/rasC⁻ cells. (B) Actin polymerization was measured in wild-type (WT) cells or rasC⁻ cells expressing Flag-RasC or -RasC^Q62L. Cells were stimulated with cAMP and lysed with Triton X-100 buffer. The Triton-insoluble pellet fraction was analyzed by SDS-PAGE and Coomassie blue staining. The amount of actin in the pellet was quantified by densitometry. The data represent mean ± SD of three independent experiments. A.U., arbitrary unit. (C) The translocation of LimEΔcoil-RFP was recorded by fluorescence microscopy with images taken every 3 s. Frames from the indicated time points after the addition of cAMP are shown. (D) The chemotactic movements to a micropipette releasing 1 µM cAMP (black dot) were recorded by time-lapse microscopy. Images from frames at 2.5-min intervals were processed to outline the cells, color coded for each time point, and overlaid. (E) The expression of cAR1 is comparable in rasC⁻ cells induced to express Flag-RasC or -RasC^Q62L. At the indicated time points after initiation of development, CHAPS-insoluble membrane fractions were prepared as described previously (Xiao et al., 1997) and probed with anti-cAR1 antibody. Bar, 10 µm.