Sc 51699

Intracellular HBV core and precore proteins and cellular p-STAT3 and suppressor of cytokine signaling (SOCS) 3 were detected using Western blot analysis. Total cellular proteins were prepared by lysing the cells on ice for 30 min in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% [V/V] NP-40, 1% [M/V] SDS, 1 mM phenylmethylsulfonyl fluoride, 10 mg/L aprotinin, 10 mg/L leupeptin). Nuclear and cytoplasmic fractionation was performed according to the manufacture's protocol (ThermoFisher Scientific, Shanghai, China). Equivalent amounts of cellular proteins (20 μg) were separated and transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). Immunoblot analysis was performed using monoclonal antibodies to p-STAT3 (sc-7993, Tyr 705) and SOCS3 (sc-51699) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal antibodies to HBV core protein (DAKO, Carpinteria, CA, USA) and enhanced chemiluminescence reagent (Invitrogen Corporation). The optical density of interesting bands was calculated using image pro plus 6.0 and showed as the ratio against that of the control of β-actin.

Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN). Crotaline (C2401) were purchased from Sigma-Aldrich (St. Louis, MO). Stattic (S7024) were purchased from Selleck (TX, USA). Human GDF11 ELISA Kit (QC-GDF11-Hu), rat GDF11 ELISA Kit (QC-GDF11-Ra), and mouse GDF11 ELISA Kit (QC-GDF11-Mu) were purchased from QCHENG BIO (Shanghai, China). Human MSTN ELISA Kit (E-EL-H1437c) and human Activin A ELISA Kit (E-EL-H0003c) were purchased from Elabscience Biotechnology (Wuhan, China). MG-132 was purchased from Tocris Bioscience (Minneapolis, MN).