Sc15404

Whole cell lysates were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting. We used antibodies against USP18 (sc-374064), AKT (sc-8312), phospho-AKT (sc-7985R), phosphotyrosine (sc-7020) from Santa Cruz at dilution 1/500; against NFκB p65/RelA (8242S), phospho(S536)-NFκB p65/RelA (3033S), insulin receptor beta-subunit (3025), JNK (9252), phospho-JNK (9251S), TAK1 (45055) and phospho-TAK-1(4531) from Cell Signalling Technology, Inc. (Danvers, MA, USA) at dilution 1/1000; against p16 (10883-1-AP) and p21 (10355-1-AP) from Proteintech (Proteintech Europe, Manchester, UK) or from BD Biosciences (San Jose, CA, USA) at dilution 1/1000; against ICAM-1 (ab2213) and VCAM-1 (ab134047) from Abcam (Abcam, Cambridge, UK) at dilution 1/1000; against tubulin, used as an index of the cellular protein content, from Sigma-Aldrich (Saint Louis, MO, USA) at dilution 1/1000. For antibodies against SIRT1 we used at first antibodies from Santa Cruz (sc15404) at dilution 1/500 then from Abcam (ab32441) at dilution 1/1000.
The activity of NFκB was measured by the ratio of the level of phosphorylation of the p65/RelA protein to the total level of p65/RelA, as measured by Western blot[36 (link)].

Briefly, liver sections (4 µm thick) were subjected to antigen retrieval and block of endogenous peroxidase activity and avidin-biotin-peroxidase method according to previous study [40 (link)]. The slides were incubated with normal serum from species producing the secondary antibody and subsequently with primary polyclonal antibodies against 4HNE (1:400, Abcam, #ab46545, Cambridge, UK), SIRT1 (1:150, Santa Cruz Biotechnology, #sc15404, Dallas, TX, USA), GRP78 (1:300, Abcam, #ab21685), SREBP1 (1:100, Santa Cruz Biotechnology, #sc8984), IL6 (1:100, Santa Cruz Biotechnology, #sc1265), p62/SQSTM1 (1:50, MBL International, Woburn, MA, USA) or monoclonal antibodies against F4/80 (1:50, Bio Rad, #MCA497GA, Segrate, Italy), Mitofusin 2 (1:200, Abnova, #H00009927, Taipei, Taiwan). All experiments were performed in triplicate. The staining intensity was expressed as arbitrary units (AU) or percentage of positive nuclei, in 20 randomly chosen microscopic fields, using an image analyzer (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, USA).