Panreac blocking buffer

Frozen liver samples were prepared for SDS–PAGE using a radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Roche). Protein samples (15 μg per lane) were separated on a 10% Bis-Tris (pH 6.6) polyacrylamide gel using NuPAGE MES SDS Running Buffer under reducing conditions (Invitrogen). After the transfer to nitrocellulose membranes, blots were blocked for 2 h in PanReac Blocking buffer (AppliChem) and incubated for 1 h at room temperature with the primary antibody against SR-B1 (NB400-101; Novus Biologicals) and against β-actin (A2228; Sigma). Membranes were incubated for 90 min in secondary horseradish peroxidase conjugated antibodies (goat anti-rabbit, Jackson Immunoresearch, 1:5,000). Detection of protein bands was performed using a luminol/para-hydroxycoumarinic acid-based chemiluminescence substrate.

Total lysates were prepared by homogenizing various tissues in RIPA buffer supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Protein samples (25 μg per lane) were separated by SDS-PAGE. Blots were blocked for 2 h in PanReac Blocking buffer (AppliChem), incubated for 1 h with appropriate primary antibody and 2 h with HRP-conjugated secondary antibody. Polyclonal rabbit antibodies directed against the C-terminal peptides of P2X4 and P2X7 were from Abcam (Cat. No. ab243734, Cat. No. ab229453). The P2X7 antibody detected two bands at around 75 kDa in BAT of Balb/c mice. The upper band was also detectable in knockout mice, indicating that as a non-specific cross-reactivity of the antibody. P2X5 rabbit polyclonal antibody was purchased from Thermo Fisher (Cat. No. PA5-41079) and the loading control γ-tubulin rabbit monoclonal antibody from Abcam (Cat. No. ab179503). The secondary antibody, goat-anti-rabbit IgG horseradish peroxidase (HRP), was purchased from Bio-Rad. Detection was performed on Amersham Imager600 using luminol and para-hydroxycoumarinic acid–based chemiluminescence substrate.