Figure 1.

siRNA-mediated depletion of PhLP inhibits Gβ expression and G protein signaling. (A) HeLa S3 cells were treated with the indicated siRNAs and then assayed after 96 h. Protein expression of PhLP, Gβ_l and lamin A/C were determined by immunoblotting. PhLP and Gβ₁ band intensities were quantified and expressed as a percentage of the lamin A/C sample. Bars and symbols in each panel represent the average±s.e. from three separate experiments. Statistical significance relative to the lamin A/C control was determined by a paired t-test (^*P<0.01). (B) Changes in intracellular Ca²⁺ in living cells were determined by measuring fluorescence from a Ca²⁺-sensitive dye using a FlexStation plate reader. Histamine (50 nM) was added at time zero and measurements were taken at the times indicated. Fluorescence data were normalized to the signal prior to histamine addition. (C) Levels of Gβ₁ and GAPDH mRNA were determined by Northern blotting. Band intensities were quantified, normalized to that of GAPDH and expressed as a percentage of the lamin A/C sample.