Cellytic mammalian tissue lysis extraction reagent

Each sample was mechanically homogenized with hand‐held pellet pestle in CelLytic™ mammalian tissue lysis/extraction reagent (Sigma‐Aldrich) prepared with a protease inhibitor cocktail (w/v 1:10, Sigma‐Aldrich). The samples were then centrifuged (10 minutes, 2500 g, 4°C), and the supernatants were collected and centrifuged again (20 minutes, 16 500 g, 4°C). Supernatant protein concentration was determined by Bradford method using Bio‐Rad protein assay (Bio‐Rad), and the samples were diluted in a reducing sample buffer (1:1, v:v) to a range of concentration of 1 μg/μL.

Streptozotocin and CelLytic™ mammalian tissue lysis/extraction reagent were purchased from Sigma Aldrich (St. Louis, MO, USA). Polyclonal rabbit anti-Oct2 antibody was purchased from AlphaDiagnostics (San Antonio, TX, USA), and polyclonal rabbit anti-PKCα and phosphorylated PKCα (p-PKCα) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal mouse anti-sodium potassium ATPase (Na⁺-K⁺-ATPase) and anti-β actin antibodies were obtained from Novus Biologicals (Littleton, CO, USA). Goat anti-mouse and rabbit immunoglobin G (IgG) horseradish peroxidase-conjugated secondary antibodies were obtained from EMD Millipore (Temecula, CA, USA). ³H-para-aminohippurate (PAH, specific activity (SA) 1 Ci/mmol) and ³H-estrone sulfate (ES, SA 50 Ci/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA, USA). ³H-1-methyl-4-phenylpyridinium (MPP⁺, SA 80 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). All other chemicals with high purity were obtained from commercial sources.