Sixty micrograms total cellular protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using a 7.5% polyacrylamide gel. Membranes were blocked with nonfat milk and then incubated overnight at 4°C with InsP3R1 or InsP3R2, Mrp2, and alpha tubulin-specific antibodies. A rabbit anti-InsP3R1 was used at a dilution of 1:3000, an affinity-purified rabbit antibody against the C-terminus of InsP3R2 isoform was used at 1:100, and a mouse antibody against the N-terminus
of the InsP3R3 was used at a dilution of 1:3000. For Mrp2 immunoblotting, an affinity-purified rabbit antibody31 was used at dilution of 1:2000; the positive control (alpha tubulin) was used at a dilution of
1:5000. Membranes were washed and incubated with peroxidase-conjugated secondary antibodies at a dilution of 1:5000. The protein-antibody conjugates were MI-503 concentration then detected by enhanced chemiluminescence (Millipore, Billerica, GSK-3 cancer MA). Confocal immunofluorescence to detect the subcellular distribution of InsP3R isoforms was performed as described previously.13, 14, 32 Briefly, frozen mouse liver sections were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100. Nonspecific binding was blocked with 1% bovine serum albumin and 5% normal goat serum. Liver sections were incubated at room temperature with primary antibody directed against specific InsP3R isoforms or against Mrp2 (Alexis Biochemicals, Farmingdale, NY) and then rinsed with phosphate-buffered saline. The specimens were then incubated with Alexa 488 secondary antibody and co-labeled with Rhodamine-conjugated Phalloidin 上海皓元 and TO-PRO-3 to facilitate recognition of hepatocyte morphology and nucleus, respectively. Images were obtained using a LSM 510 confocal microscope (Zeiss, Thornwood, NY) by excitation at 488 nm and observation at 505 to 530nm to detect Alexa 488. Rhodamine phalloidin was excited at 543 nm and observed at 585 nm and TO-PRO3 was detect by using a 647 nm laser
line and observed above 665 nm. Cytosolic Ca2+ was monitored in isolated mouse hepatocytes and HepG2 cells. CMFDA secretion was monitored in the canalicular spaces between mouse hepatocytes in collagen sandwich culture by time-lapse confocal microscopy as described previously.13, 33 Briefly, hepatocytes were incubated with either Fluo-4/AM (6 μM) for 30 minutes at 37°C for Ca2+ imaging or Cell tracker Green CMFDA (1 μm) to monitor organic anion secretion. Cover slips seeded with the cells were transferred to a custom-built perfusion chamber on the stage of an LSM 510 confocal microscope (Zeiss, Thornwood, NY), and the cells were then perfused with 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid-buffered solution. Cells were excited at 488 nm and observed at 505 to 550 nm. In most experiments, 40× or 63× objectives were used to observe the cells.