Fatp2

Routine Western Blotting procedures were used to detect the total expression of fatty acid transport proteins: FATP2, FATP5 (Santa Cruz Biotechnology, USA), FAT/CD36, FABPpm (Abcam, UK), ABCA1 (Thermo Scientific, USA), MTP (Santa Cruz Biotechnology, USA), ACBP and L-FABP (Abcam, UK) as well as the proteins directly involved in lipogenesis (FAS; Cell Signaling, USA), oxidation pathway (CPT 1; Santa Cruz Biotechnology, USA) and lipid metabolism (PPARα, LXR, SREBP1c, pAMPK; Cell Signaling, USA) as previously described in details by Konstantynowicz-Nowicka et al. [17 ]. All the antibodies used in our procedures were monoclonal except for FATP2 and FAT/CD36. Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk, the membranes were immunoblotted with primary antibodies of interest and incubated with secondary antibodies labeled with horseradish peroxidase (HRP). Obtained protein bands were quantified densitometrically using a ChemiDoc visualization system (Bio Rad, Warsaw, Poland). Equal protein loading was controlled by Ponceau S staining. The expression of all the proteins was standardized to the GAPDH (Santa Cruz Biotechnology, USA) expression and the control was set as 100%.

Liver cell fractionation was performed using a simplified method adapted from Jiang and colleagues (Liu et al, 2011). Liver organelle fractionation was conducted via density‐based separation (Cox & Emili, 2006). RNA was extracted from tissues using Qiazol and cDNA synthesised using the First Strand cDNA synthesis kit (Fermentas, DEU). Quantitative PCR was conducted using Taqman master mix and Taqman primer‐probe assays (Life Technologies, DEU). Tissue protein extraction and immunoblotting was performed using standard methods using GADD45β (sc‐8776), FATP2 (sc‐161311), FABP1 (sc‐50380), HNF4a (sc‐6556), BCKDE1A (sc‐67200), NTCP (sc‐98485) and ARG1 (sc‐21050) (Santa‐Cruz Biotechnology, DEU); LC3B (2275), pT39‐S6K1 (9205), p‐ERK (9101), p‐p38 (9211), p‐eIF2a (9721) and GRP78 (3183) (Cell Signaling Technologies, USA); CD36 (AF2519, RnD Systems, USA); and the housekeeping protein VCP (ab11433, Abcam, UK) antibodies. Immunoprecipitation was conducted using anti‐FLAG (A2220, Sigma‐Aldrich, DEU) and anti‐HA (A2095, Sigma‐Aldrich, DEU) agarose from tissue lysates using standard protocols.