Heterologous Expression of GLUT4 and AQP7

Rat GLUT4 with N‐terminal poly‐histidine tag and human AQP7 (Aquaporin 7, as a control in far dot western blot) with C‐terminal His‐tag were cloned into the double‐deletion strain Pichia pastoris GS115 aqy1Δ::HIS4 agp1Δ::NatMX with no endogenous aquaporins and aquaglyceroporins present [21 (link)]. The yeast cells with GLUT4 and AQP7 construct were cultured as previously described [22 (link)]. Harvested cells were lysed with high‐pressure homogenization apparatus (Biox AB, Onsala, Sweden), subsequently, membranes were isolated by ultra‐centrifugation at 145 000 x g for 90 min at 4 °C and solubilized in 20 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, EDTA‐free protease inhibitor cocktail (Merck, Darmstadt, Germany) and 1% decyl β‐D‐maltopyranoside (DM, Anatrace, Maumee, OH, USA) for 1 h at 4 °C. Homogenate was clarified by centrifugation at 145 000 x g for 30 min, and subjected to western blot analysis. In brief, the PVDF membrane was blocked in 2% fish gelatin before incubating with GLUT4 antibody [18 (link)] and corresponding HRP conjugated secondary antibody (Invitrogen, Waltham, MA, USA) to identify GLUT4 protein in the lysate. Finally, the signal was recorded with Odyssey Fc gel‐scanning system (LI‐COR, Lincoln, NE, USA).