Nf κβ

Protein was isolated in situ, and western analysis performed as previously described [27 (link)]. Blots were probed using antibodies directed to Claudin-1, Occludin-1, ZO-1 (Thermo Fisher Scientific, North Ryde, Sydney, NSW, Australia), LC3, poly (ADP)-ribose polymerase (PARP), Sequestosome (Cell Signaling Technology, Boston, MA), Bcl2, NF-κβ (Santa Cruz Biotechnology, Dallas, TX) and β-actin (Sigma-Aldrich) followed by matching horseradish peroxidase-conjugated secondary antibodies (R&D Systems, MN, USA). Detection was performed using ECL Prime chemiluminescent substrate (GE Healthcare, Buckinghamshire, UK). Densitometry of histogram analyses was performed using Multi Gauge software (V3.1 Fugifilm, Tokyo, Japan). Density scores were normalized to both β-actin and the untreated control, and analyzed using a bootstrapped gamma regression model (log link) and stratified by replicate. The statistical analysis was performed using R statistical software (release 3.2.3) and results expressed as relative abundance.

The proteins suspension of liver tissues were obtained using a total protein extraction kit (APPLYGEN, Beijing, China) and protein concentrations were determined by the BCA protein assay kit while using BSA as a standard. Subsequently, standard western blot procedures were followed, as described elsewhere [41 (link)]. β-actin was used as an endogenous control and blots were quantified by using Image J Software (NIH, Bethesda, MD, USA). The primary and secondary antibodies used for western blot (AMPK, AMPK-p, p-ACC, ACC, CD68, and β-actin) were purchased from Abcam (Cambridge, UK) and other antibodies (CaMKKβ, NF-κβ, TGF-βR1, PPARα, SMAD2/3-P, and SMAD2/3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Standard immunoblotting and immunehistochemistry protocols were carried out. Antibodies used were AR (N-20, Santa Cruz, CA, USA). PTen, AKT, p-Akt, NFκβ, IFNγ, HIF1α and STAT3 were obtained from Santa Cruz. CD3 was from DAKO (Santa Clara, CA USA) and CD45R was from Biolegend (San Diego, CA, USA). The Vectastain avidin–biotin complex (Vector Labs, Peterborough, U.K.) was used for detection, using diaminobenzidine chromogenic substrate. Negative controls were included lacking primary antibody. Images were captured using a Leica DM1000 microscope.