Midi transfer packs

All reagents were obtained from Life Technologies (Paisley, UK) unless otherwise stated. Fetal bovine serum (FBS) and HS were purchased from Seralab (Haywards Heath, UK). DC protein assay, TGX gels and midi transfer packs were all purchased from Bio‐Rad (Hemel Hempstead, UK). BLUeye Prestained Protein Ladder was purchased from Geneflow (Lichfield, UK). LONG^® R3 insulin‐like growth factor (IGF)‐1 (human) was purchased from Sigma‐Aldrich (Gillingham, UK). Anti‐pan Akt and anti‐p‐Akt (serine 473) were purchased from Cell Signaling Technologies (MA, USA), anti‐β‐actin from Abcam (Cambridge, UK) and anti‐apoB from Santa Cruz Biotechnology (CA, USA). Amicon Ultra Centrifugal Filter units were purchased from Millipore (Watford, UK). RNEasy^® Plus mini kit was purchased from QIAGEN Sciences (Manchester, UK) and KAPA PROBE FAST qPCR MasterMix (2X) from Kapa Biosystems (London, UK). Taqman probes were purchased from Applied Biosystems (Hemel Hempstead, UK). TAG, lactate and 3‐hydroxybutyrate (3‐OHB) assays were from Instrumentation Laboratory UK (Cheshire, UK). Heavy water (²H₂O) was purchased from CK Isotopes (Ibstock, UK) and gas chromatography (GC) standards were from Sigma‐Aldrich (Gillingham, UK).

Protein was isolated from cells as previously described (38 (link)) and was quantified using the Pierce Microplate BCA protein assay kit - reducing agent compatible (Thermo-Scientific), run on Bio-Rad Any-kDa Mini-Protean TGX precast gels, and transferred to nitrocellulose membranes in the Bio-Rad Midi Transfer Packs using the Bio-Rad Trans-Blot turbo blotting system. The membranes were blocked 1 h in 5% milk, before being probed overnight at 4 °C with SOX9 antibody (1:800, Abcam catalog no. ab26414). Secondary antibody was applied for 2 h following wash steps at the following dilutions: goat α-rabbit (1:4000, Abcam ab97069). Preconjugated β-actin-HRP (1:40,000, Sigma-Aldrich catalog no. A3854) was applied for 20 min and used as loading control. Western blot densitometry analysis was done using ImageJ.