Glycerol

Gelatin powder (17009-01, Kanto Chemical) and glycerol (17029-00, Kanto Chemical) were purchased from a supplier. The Gelatin powder (Gel), glycerol (Gly), and distilled water (Wat) were mixed in a mass ratio of Gel:Gly:Wat = 2:1:8, placed in a beaker, and left to stand for 10 min to soften the Gelatin powder. The solution was then stirred at 200 rpm at 80°C for 30 min to completely mix the ingredients. Subsequently, the mixture was poured into a set of molds that have internal dimensions of W 110 mm × L 110 mm (Figure 1A). Each mold was filled with 30 g of the solution. After removing the air bubbles by spraying ethanol, the samples were cured at 40°C for 2 h in an oven to form films with an average thickness of ∼0.5 mm. The films were then carefully removed from the molds and stored in a humidity chamber (WET-297-AHU, Tolihan) at room temperature (∼24°C) and 67% relative humidity (RH).

IEF and SDS-PAGE were performed as described by Shen et al. [15 (link)]. The BioRad Protean IEF Cell was first used to rehydrate the 18 cm immobiline^TM dry strips (pH 4–7) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a 300 µL rehydration buffer containing 200 µg of protein lysates made from mock and PEPE2-treated cells for 16 h at 20 °C. After rehydration, the proteins were concentrated at 20 °C under 50, 100, 200, 500, 1000, 5000, and 8000 V, respectively, with 81,434 voltage hours. Then, an equilibration buffer [2% (w/v) SDS (aMResco, Solon, OH, USA), cotaining 30% (v/v) glycerol (Kanto Chemical, Portland, OR, USA), and 6 M urea (aMResco, Solon, OH, USA)] with 2% (w/v) DTT (USB Corcorporation, Cleveland, OH, USA) was implemented to equilibrate the gel strips for 15 min. Afterwards, the DTT-equilibrated strips were incubated for 15 min with an equilibration buffer comprising 5% (w/v) iodoacetamide (aMResco, Solon, OH, USA). Then, the strips were put above a 12.5% (w/v) polyacrylamide gel sealed with 0.5% (w/v) agarose (aMResco, Solon, OH, USA) gel, and various proteins were resolved at 420 V using BioRad Protean IIxi until bromophenol blue run near the bottom.

Isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were conducted as described previously [45 (link)]. The 18-cm immobibline dry strips (pH 4–7) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were rehydrated within the BioRad Protean IEF Cell for 16 h at 20 °C with 300 μL rehydration buffer including 200 μg protein lysates respectively prepared from Sh-Gal-1(+120) and Sc-Gal-1(+120) T24 cells. Then, the proteins were concentrated at 20 °C at 50, 100, 200, 500, 1000, 5000, and 8000 V, respectively, with a total of 81,434 voltage-hours. After isoelectric focusing, the gel strips were equilibrated in the equilibration buffer (6 M urea, 30% (v/v) glycerol (Kanto Chemical, Portland, OR, USA), 2% (w/v) SDS (aMResco, Solon, OH, USA)) containing 2% (w/v) DTT for 15 min and then in the equilibration buffer containing 5% (w/v) iodoacetamide (aMResco, Solon, OH, USA) for a further 15 min. The equilibrated gel was loaded onto the top of a 12.5% (w/v) polyacrylamide gel and sealed with 0.5% (w/v) agarose (aMResco, Solon, OH, USA) and the proteins were separated at 420 V using BioRad Protean IIxi until bromophenol blue reached the bottom of the gel.