Fig. 3. PLCβ1a and PtdIns(4,5)P₂. (A) HeLa cells were stimulated with histamine (100 µM, arrow) after overnight incubation with 1 µM PMA. The red trace depicts the mean normalized fluorescence intensity of GFP-PLCβ1a; the black trace depicts the mean normalized fluorescence intensity of the Fura Red dye in a region of interest in the cytosol (n=7; the error bars depict standard error). The Fura Red dye was excited at 488 nm and detected with a long-pass 650-nm filter. Therefore, an increase in intracellular Ca²⁺ correlates with a decrease in fluorescence intensity. For the exact settings used, see Materials and Methods. (B) HeLa cells expressing RFP-PH1. Scale bar: 10 µm. (C) HeLa cells coexpressing PLCβ1a (red trace), PH1 (black trace) and bradykinin type 2 receptor (B2R) were first stimulated with histamine (100 µM; at t=15 seconds, first arrow) and then with bradykinin (1 µM; at t=115 seconds, second arrow).