Plcδ1

Hepatocytes were grown on collagen-coated confocal dishes and fixed with 3.8% paraformaldehyde for 20 min, after which they were washed three times with ice-cold PBS. To permeabilize, cells and nuclei were treated with 0.25% Triton X-100 in PBS for 10 min. After blocking with 3% BSA, 0.25% Triton X-100, and PBS at RT for 1 h, samples were incubated overnight at 4 °C with the indicated antibodies: TRPM2 (Novus, NB110-81601), CD38 (Thermo Fisher Scientific, 14-0381-85), PKCδ (Santa Cruz, sc-8402), Cx43 (Santa Cruz, sc-13558), PLCδ1 (Santa Cruz, sc-365811), PLCδ3 (Santa Cruz, sc-514912), or Lamin B1 (Santa Cruz, sc-6216). Alexa Fluor-conjugated secondary antibodies (546 donkey anti-rabbit antibody, Thermo Fisher Scientific, A10040; 555 donkey anti-goat antibody, Thermo Fisher Scientific, A-21432; 488 donkey anti-rat antibody, Thermo Fisher Scientific, A-21208; 488 donkey anti-mouse antibody, Thermo Fisher Scientific, A-21202; or 488 donkey anti-rabbit antibody, Thermo Fisher Scientific, A-21206) were incubated at 1:500 dilutions in the presence of 1% BSA at RT for 1 h. The nuclei were stained with DAPI (Thermo Fisher Scientific, 62248). Cells and nuclei were visualized with a Zeiss LSM510 Axiovert 200 M laser-scanning confocal microscope.

Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 10% skim milk and incubated with the antibodies against E-cadherin (BioLegend), β-catenin (BioLegend), claudin-1 (Santa Cruz Biotechnology), PARD3 (Merck Millipore), Akt (Cell Signaling Technology), phospho-Akt (Ser473) (Cell Signaling Technology), PLCδ1 (Santa Cruz Biotechnology), N-cadherin (BD Biosciences), and β-actin (Sigma-Aldrich), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). Images were captured using LuminoGraph I (ATTO, Tokyo, Japan).