Magicmark

Thirty-forty micrograms of lysate proteins for each sample together with the molecular weight Magic Mark (Invitrogen, Carlsbad, CA, USA) were subjected to 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation (Bis-Tris Plus BOLT, Invitrogen) and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Burlington, MA, USA). The membranes were blocked in 5% skim milk and incubated overnight with the following specific primary antibodies: rabbit anti-p16 (N-20, sc-467, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit COX-2 (160126, Cayman Chemical, Ann Arbor, MI, USA), rabbit anti-NFkB (GTX102090, GeneTex, Irvine, CA, USA), mouse anti-α smooth actin (A2547, Sigma-Aldrich Chemicals, Italy), rabbit anti-Glucose 6 Phosphate Dehydrogenase (GAPDH) (14C10, Cell Signaling) followed by the suitable HRP-conjugated secondary antibodies (Sigma-Aldrich Chemicals). All resulting immunocomplexes were visualized with an enhanced chemiluminescence ECL detection system (GE Healthcare, Milano, Italy) and quantified by ImageJ software (NIH, Bethesda, MD, USA). Each density measure was normalized by using the corresponding GAPDH level as an internal control.

Thirty‐forty micrograms of lysate proteins for each sample together with the molecular weight Magic Mark (Invitrogen, Carlsbad, CA, USA) were subjected to 4–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis separation (Bis‐Tris Plus BOLT, Invitrogen) and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Burlington, MA, USA). The membranes were blocked in 5% skim milk and incubated overnight with the following specific primary antibodies: rabbit anti‐lamin B1 (Cell Signalling), mouse anti‐Tubulin (Sigma) followed by the suitable HRP‐conjugated secondary antibodies (Sigma‐Aldrich Chemicals). All resulting immunocomplexes were visualized with an enhanced chemiluminescence ECL detection system (GE Healthcare, Milano, Italy) and quantified by ImageJ software (NIH, Bethesda, MD, USA).

Western blotting was performed mainly as described previously [36 (link)]. Briefly, cell lysates in reducing sample buffer were electrophoresed in SDS-12% polyacrylamide gels under reducing conditions, in parallel with protein molecular mass standards (MagicMark^™, Invitrogen). Subsequently, proteins were transferred onto nitrocellulose membranes (Amersham Protran, GE Healthcare). Membranes were blocked with PBS containing 0.05% Tween 20 and 2% ECL Prime Blocking Agent (GE Healthcare), and probed 1h with primary antibodies directed against HCV core protein (C7-50, Alexis Biochemicals), HCV E2 glycoprotein (AP33, kindly provided by A. H. Patel, MRC Virology Unit, Institute of Virology, Glasgow, UK), HCV NS3 protein (8 G-2, Abcam), or calnexin (C4731, Sigma-Aldrich). Excess antibody was removed with five washes, and the second-step antibody (DyLight 800 labeled goat anti-mouse or DyLight 680 labeled goat anti-rabbit, KPL) diluted 1:15,000 in blocking buffer was allowed to bind for 1 h. After several washes, protein bands were visualized using LI-COR-Odyssey infra-red scanner (LI-COR).

Western blotting was performed using standard procedures, and equal amounts of total protein were electrophoresed on 4–20% Tris-Glycine gels and MagicMark (Invitrogen, Carlsbad, CA, USA). Antibodies used are FOXO3A and phospho-FOXO3A-Ser318/321 (numbers 9467 and 9465, resp., Cell Signaling Technology); SGK1 (number 3272, Cell Signaling Technology) and β-actin were used as control.

Anti‐P‐Ser‐473 Akt (#9271) and anti‐AKT antibodies (#9272) were from Cell Signaling Technology. The phosphospecific antibody to the α₁‐isoform of the Na‐K pump that is phosphorylated at Ser⁹³⁸ (anti‐P‐Ser⁹³⁸) of the rat Na‐K‐ATPase α₁‐isoform was developed by Cell Signaling Technology and previously characterized (Massey et al., 2016 (link)). The antibody against the α‐subunit of sheep kidney Na^+/K⁺ ‐ATPase (M8‐P1‐A3) was purchased from Sigma–Aldrich. Secondary antibodies were from Jackson ImmunoResearch: donkey anti‐mouse (# 715‐035‐150) and anti‐rabbit (# 711‐035‐152) both coupled to horseradish peroxidase (HRP). H‐89 was from LC Laboratories. SDS‐PAGE reagents were from Fisher Scientific (Hanover Park); acrylamide was from Bio‐Rad Laboratories; polyvinylidene difluoride was from Millipore; KPL chemiluminescence reagents were from Insight Biotechnology; phosphatase inhibitors (microcystin and okadaic acid) were from Axxora; the molecular weight marker MagicMark was from Invitrogen. CPT‐2Me‐cAMP (CPT) was from Tocris. All other reagents were from Sigma‐Aldrich.

Frozen liver and EWAT were homogenized on ice in RIPA Buffer (Sigma Chemical, Inc.) (10:1 buffer to QUAD mass and 5:1 buffer to EWAT mass ratios) containing protease and phosphatase inhibitor cocktails (Halt Protease/Halt Phosphatase, Thermo Fisher). Homogenates were rotated at 4°C for 1 h and centrifuged at 9,000 x g for 30 min at 4°C. The protein concentrations of the supernatants were determined by the BCA method (Pierce, Inc), and equal amounts of protein were subjected to SDS-PAGE along with molecular weight standards (Bio-Rad, Hercules, CA and Magic Mark, Invitrogen). Proteins were then electrophoretically transferred to Immobilon membranes and immunoblotted with Cidea and Cidec antibodies. Light generated by the alkaline phosphatase conjugated secondary antibody and CDP-Star reagent was detected using a digital imaging system (UVP, Upland, CA). To account for gel loading differences, all immunoblots were stripped and re-probed with a ß-tubulin antibody. Relative signal intensities of immunoreactive bands were determined using Total Lab software (Nonlinear, Inc., Durham, NC) or GraphPad Prism and ImageJ. All data were normalized to ß-tubulin and expressed as a percentage of LFD-Vehicle. The ß-actin antibody was from Cell Signaling, the Cidea antibody was from Sigma, and the Cidec antibody was a kind gift from Cynthia Smas, Toledo, Ohio.