Sc 45045

ECFCs were lysed in radioimmunoprecipitation assay (RIPA) buffer in the presence of protease (Complete Ultra, Roche, Penzberg, Germany) and protein phosphatase inhibitors (sc-45045 and sc-45065, Santa Cruz Biotechnology, Santa Cruz, US). Proteins were separated by SDS-PAGE and immunolabelled using specific antibodies for ERK1/2 (sc-94, Santa Cruz Biotechnology) and phospho-ERK1/2 (9101S, Cell Signaling). Extracts from cell-free hPLG-coated wells were utilized as negative controls, which highlighted the presence of dephosphorylated ERK1/2 from platelets. This did not interfere with the analysis of ERK1/2 phosphorylation. Blots were stained with IRDye 800CW or IRDye 680RD secondary antibodies and scanned with a LI-COR Odyssey CLx infrared imaging system. Densitometric analyses were performed using Image Studio Lite v5.0.21 (LI-COR, Lincoln, US).

Liver tissues were homogenized in lysis buffer (0.1% w/v SDS, 0.1% w/v sodium deoxycholate, 1% v/v Triton X-100 in PBS) with a protease inhibitor cocktail 1:200 (v/v) (P9599; MilliporeSigma, Madrid, Spain) and a phosphatase inhibitor cocktail 1:100 (v/v) (SC-45045; Santa Cruz Biotechnology, Dallas, TX, USA) using a Bullet Blender (Next Advance, Averill Park, NW, USA). Homogenates were sonicated and centrifuged 12,000 g/10 min/4 °C. After protein concentration was determined by the bicinchoninic acid assay [29 (link)], samples were heated at 95 °C for 5 min in Laemmli sample buffer (BioRad, Hercules, CA, USA). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Samples were immunoblotted using the primary antibodies rabbit anti-AKT (ref. 9272s, Cell Signaling Technology, MA, USA), Ser473 rabbit anti-pAKT antibody (ref. 4060s, Cell Signaling Technology, MA, USA), rabbit anti-AMPKα antibody (ref. 2532L, Cell Signaling Technology, MA, USA), Thr172 rabbit anti-pAMPKα antibody (ref. 2535s, Cell Signaling Technology, MA, USA), rabbit anti-PPARα antibody (ref. ab3484, Abcam, Cambridge, UK) and mouse anti-actin (A5441, Sigma-Aldrich, San Luis, MO, USA) at 1:1000 dilution. After incubation with a secondary antibody, bands were detected by ECL (PerkinElmer, Waltham, MA, USA) and quantified using ImageJ (National Institutes of Health, USA).