Effects of CaSR, signal transduction inhibitors, and trypsinization on Ca_o²⁺-mediated Ca_i²⁺ changes. (A) PBMCs were treated with U73122 (10 μM) and its negative control U73343 (10 μM), genistein (50 μg/mL), or DMSO (2 μL/mL) for 30 minutes prior to adding Indo-1 AM and CD19 PE antibodies. Calcium flux data were obtained upon the addition of 500 μM CaCl₂ () in B cells over a 6-minute period. (B) Isolated B cells were cultured in PBS with or without genistein or U73122 for 30 minutes prior to adding CaCl₂ (500 μM) for 2 minutes followed by cell lysis. Samples were then electrophoresed and immunoblotted for phosphorylated (P)–tyrosine, P-serine PKC substrate, and β-actin (loading control). (C) B cells were treated with trypsin or PMA (16 nM) for 30 minutes and 4 hours, respectively, in PBS prior to calcium flux analysis. (D,E) RNA and protein were extracted from C6 cells, isolated monocytes (Mo's), and B cells from 2 different donors (B1, B2) prior to one-step RT-PCR (D) or immunoblotting (E) for CaSR expression. (F) PBMCs or rat brain C6 cells were treated with NPS2390 (20 μM) for 30 minutes prior to calcium flux analysis. (G) B cells were treated with Syk inhibitor (20 μM) for 30 minutes prior to calcium flux analysis in response to 10 μg/mL anti-Ig or 500 μM CaCl₂. Data are representative of 3 different samples.