Mini protean tetra cell electrophoresis equipment

For direct protein extraction, the samples were mixed with the denaturing and reducing sample buffer (125 mmol/L Tris-HCl pH 6.8, 4% SDS, 20% v/v glycerol, DTT 50 mg/mL), boiled at 100 °C for 5 min, and centrifuged. Then, SDS-PAGE electrophoresis was performed using the method of Schägger–von Jagow [25 (link)]. The PageRuler Prestained Protein Ladder and Spectra Multicolor Low Range Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) were used as molecular weight protein markers. Gels were run in the Mini PROTEAN Tetra Cell electrophoresis equipment (Bio-Rad Laboratories, Hercules, CA, USA) and stained with Coomassie Brilliant Blue R-250.

PGM2p-GFP fusion protein content was analyzed using quantitative western blotting, as indicated previously [13 (link),14 (link)]. Gene deletion mutant strains in the PGM2p-GFP background were cultured in media either containing or lacking LiCl to evaluate the protein levels of PGM2. Protein concentration was determined using the Bradford Protein Assay (BSA). Using Mini-PROTEAN Tetra cell electrophoresis equipment (Bio-Rad^®, Mississauga, ON, Canada), 50 µg of total extracted protein was run on a 10% SDS-PAGE gel. Trans-Blot Semi-Dry Transfer (Bio-Rad^®, Mississauga, ON, Canada) was used to transfer protein bands onto a 0.45 m nitrocellulose membrane. A mouse monoclonal anti-GFP antibody (Santa Cruz^®) was used to detect PGM2p-GFP protein levels, and a mouse monoclonal anti-PGK1 antibody was used to assess PGK1 protein levels (internal control). At least three technical and biological replicates were used in each experiment.

Extraction was performed using a mixture containing 125 mM Tris-HCl pH 6.8, 4% SDS, 20% v/v glycerol, and DTT 50 mg/mL at 100 °C for 5 min. The Schägger-von Jagow method [39 (link)] with protein molecular weight standards in the range of 10–180 kDa (PageRuler Prestained Protein Ladder, Thermo Scientific, Waltham, MA, USA) was used to perform SDS PAGE electrophoresis. Gels were run in Mini Protean Tetra Cell electrophoresis equipment (Bio-Rad, Hercules, CA, USA) and stained with Coomassie Brilliant Blue R-250.