Chemidoc visualization system

The expression of proteins directly involved in lipogenesis (Fas; Cell Signaling, Beverly, MA, USA), oxidation pathway (Cpt1, β-had; Santa Cruz Biotechnology, Dallas, TX, USA) and the process of desaturation and elongation (Elovl3, Elovl6, Scd1; Santa Cruz Biotechnology) as well as fatty acid exporting proteins: Abca1 (Thermo Scientific, Waltham, MA, USA) and Mtp (Santa Cruz Biotechnology) were detected by routine Western Blotting as previously described in details by Konstantynowicz-Nowicka et al. [28 (link)]. Briefly, protein concentration was determined using bicinchonic acid method (BCA) with bovine serum albumin (BSA) as a standard. Signals obtained by immunoblotting were quantified densitometrically using a ChemiDoc visualization system (Bio Rad, Warsaw, Poland). Equal protein loading was confirmed using Ponceau S staining. The expression of all the proteins was standardized to the Gapdh (Santa Cruz Biotechnology) expression and the control was set at 100%.

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%.

The cells were lysed in 2× lysis buffer containing 200 mM Tris-HCl, protease inhibitor cocktail (Roche, Pleasanton, CA, USA), 400 mM β-mercaptoethanol (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 4% sodium dodecyl sulfate (SDS; Serva), 0.01% bromophenol blue, and 40% glycerol (PanReac, Barcelona, Spain) and incubated at 95 °C for 1 min. Proteins were separated using 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred from the gel to PVDF membranes by a semi-wet approach using Trans-Blot^® Turbo™ RTA Mini LF PVDF TransferKit (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% milk on tris-buffered saline containing 0.1% Tween (TTBS) for 1 h at room temperature, then stained overnight with primary antibodies against GAPDH, GRP78, LC3B, involucrin, filaggrin, and subsequently with HRP-conjugated secondary antibodies (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Target proteins were visualized by Novex ECL Kit (Invitrogen, Waltham, MA, USA) in the ChemiDoc™ visualization system (Bio-Rad Laboratories, Inc.). Optical density of the protein bands was determined using ImageLab Software (Bio-Rad Laboratories, Inc.).