Western blot and deglycosylation

Samples were heated at 95°C for 5 min in 2× SDS and 20× reducing agent (Bio-Rad) and loaded into 10% Bis/Tris Criterion XT gels (Bio-Rad). The electrophoretic field was set at 200 V for 35 min followed by transfer to nitrocellulose membranes at 240 mA for 45 min and blocked in 5% (wt:vol) nonfat milk in Tris-buffered saline for 4 h before incubation overnight with primary antibodies. Membranes were independently blotted for Mfsd2a (1:1000; PA5-21049) and Glut1 (1:1000; PA1-46152) (Thermo Fisher Scientific). Blots were developed with the use of Amersham ECL Western Blotting Detection Reagent (GE Healthcare Biosciences) and visualized on a ChemiDoc XRS+ (Bio-Rad), and analyses were performed using Image Lab software 3.0 (Bio-Rad). Membranes were stripped (Re-blot Plus; Thermo Fisher Scientific) and reprobed with β-actin antibody (1:2000; AC-40) (Sigma-Aldrich). Samples sets were run in duplicate, and the respective protein expressions were normalized to the average of the 10% lard group normalized to β-actin.

Peptide:N-Glycosidase F (PNGase F) assay (New England BioLabs, Inc.) catalyzes the cleavage of an internal glycoside bond from N-linked glycoproteins. Samples were mixed with glycoprotein denaturing buffer (10×) and deionized H₂O, heated at 100°C for 10 min, then centrifuged at 4°C for 10 s at 2000 × g, followed by addition of 2 μL GlycoBuffer-2 (10×) and 2 μL 10% NP-40, and mixed on a vortex for 10 s. Then, 3 μL PNGaseF or 3 μL dH₂O (control) was added and incubated in a dry heater for 24 h followed by Western blot analysis as described above.