Appl1

Proteins from total liver or cellular lysates or immunoprecipitated were separated by SDS– polyacrylamide gel electrophoresis (SDS–PAGE), and probed with different primary antibodies as specified in each figure legend. The specific signals were amplified by addition of horseradish peroxidase-conjugated secondary antibodies and visualized with enhanced chemiluminescence (ECL from Millipore). Western blotting images were processed using a ChemiDoc XRS digital imaging system with Quantity One 1-D analysis software (Bio-Rad Laboratories, Inc.).
Antibodies from Cell Signaling Technology (working dilution 1:1,000) were the following: phospho-Akt (Ser473) (#4060), phospho-Akt (Ser308) (#13038), phospho-Akt2 (#8599) phospho-GSK3 (Ser9)(#9327), phospho-FoxO1-3 (Thr24/32) (#9464), phospho-p70S6K (Thr389) (#9234), phospho-Glycogen Synthase (Ser641) (#3891), total Akt1 (#2967), total Akt2 (#3063, #5239), total Glycogen Synthase (#3893), total FoxO3 (#12829), total GSK3 (#12456), total p70S6K (#2708), APPL1(#3858), Rab5(#2143) (#3547), Rab7(#2094), Myc-Tag (#2272). Antibody to Glut2 (working dilution 1:1,000) was from Santa Cruz Biotechnology Inc (sc-9,117). Original gel images are shown in Supplementary Fig. 9.

The antibodies against PKCζ, pAKT, AKT, TRAF6, APPL1, pERK, ERK, pSmad2, Smad2, P65, LaminA, were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA) and used at 1:1000 dilution for Western blotting. HAS2 (Santa Cruz, CA, USA) was used at 1:200, and β-actin and β-tubulin (Sigma, St. Louis, MO, USA) was used at 1:1000 for Western blotting. Antibodies against LYVE-1, GFP, F4/80 (Abcam, UK), Ki67 was used at 1:500 dilution (Dako, Denmark), Alexafluor 488 and Alexafluor 555 (Invitrogen, Oregon, USA) were used for immune-staining. 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was from Merck, (Darmstadt, Germany). Pefabloc was from Roche (Mannheim,Germany), and PageRuler prestained protein ladder was from Thermo Scientific.

The utilized antibodies for immunohistochemistry and immunocytochemistry were APPL1 (#3858 Cell Signaling, Danvers, MA, USA; antibody dilution: 1:100), APPL2 (#14294-1-AP Thermo Fisher Scientific, Waltham, MA, USA; antibody dilution: 1:200), α-p-p38-MAPK (T180/Y182) (#4511 Cell Signaling, Danvers, MA, USA; antibody dilution: 1:100), NF-κB (p65) (#8242 Cell Signaling, Danvers, MA, USA; antibody dilution: 1:500), GLUT4 (#sc-53566 Santa Cruz Biotechnology, Dallas, TX, USA; antibody dilution: 1:200), p-AS160 (T642) (#MBS002191 MyBiosource, San Diego, CA, USA; antibody dilution: 1:100), p-MEK (S189) (#ab194809 Abcam, Cambridge, UK; antibody dilution: 1:200), anti-IgG Rabbit secondary antibody (#31460 Cell Signaling, Danvers, MA, USA) and anti-IgG Mouse secondary antibody (#31430 Cell Signaling, Danvers, MA, USA). In this study, other reagents for stimulated cells in in vitro assays were used: an adiponectin agonist called AdipoRon (AdipoR agonist, 2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]-acetamide, #SML0998, Sigma-Aldrich (Merck), Darmstadt, Germany); a human recombinant protein called TNFα (tumor necrosis factor alpha, #H8916, Sigma-Aldrich, MO, USA) and metformin (1,1-Dimethylbiguanide hydrochloride, #1115-70.4, Sigma-Aldrich (Merck), Darmstadt, Germany).