Goat anti rabbit igg horseradish peroxidase hrp

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.

Primary antibodies against p21, CDK2, Cyclin D1, CDK6, Mcl-1, Bcl-xL, Bcl-2, Bax, Poly (ADP-Ribose) Polymerase (PARP), phospho-AKT (Ser473), AKT, phospho-mTOR (Ser2448), mTOR, phospho-70S6K (Thr421/Ser424), 70S6K and LC3B were purchased from Cell Signaling Technology (Beverly, MA, United States). An antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The secondary antibodies, including goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP, were obtained from Bio-Rad (United States). Dimethyl sulfoxide (DMSO), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3-methyladenine (3-MA), chloroquine (CQ) LY294002, bafilomycin A1 (Baf-A1) and rapamycin (Rapa) were purchased from Sigma Company (St. Louis, MO, United States). IATL with the purity≥98% as determined by high performance liquid chromatography was bought from Chengdu Must Bio-Technology Co. Ltd. (Chengdu, China). The chemical structure of IATL is shown in Figure 1A.