Fusion fx5 xt

Western blot analysis was used to investigate the protein content for Pgm2p-GFP fusion protein. Deleted mutant strains with GFP-tagged PGM2 were grown in media treated with and without LiCl to investigate protein levels of PGM2. Protein extraction was performed as described by Szymanski et al. [75 (link)]. Bicinchoninic acid assay (BCA) was performed to estimate protein concentration as described by the manufacturer (Thermo Fisher^®, Ottawa, ON, Canada). Equal amounts of total protein extract (50 μg) were loaded onto a 10% SDS-PAGE gel, run on Mini-PROTEAN Tetra cell electrophoresis apparatus system (Bio-Rad^®). Proteins were transferred to a nitrocellulose 0.45 μm membrane via a Trans-Blot Semi-Dry Transfer (Bio-Rad^®). Mouse monoclonal anti-GFP antibody and anti-Pgk1 (Santa Cruz^®) were used to detect protein levels of Pgm2p-GFP and Pgk1p, respectively. Immunoblots were visualized with chemiluminescent substrates (Bio-Rad^®) on a Vilber Lourmat gel doc Fusion FX5-XT (Vilber^®). Densitometry analysis was carried out using the FUSION FX software (Vilber^®, Collégien, France). Experiments were repeated at least three times. T-test analysis (p-value ≤ 0.05) was used to determine statistically significant results.

In order to observe the post-exercise activity and long-term exercise induced adaptation of HSL, protein expression and phosphorylation of VAT, SCAT, and isolated adipocytes were tested after epinephrine stimulation (see details in in vitro Lipolysis Test part). Chopped tissue and isolated cells were homogenized with RIPA buffer (Solarbio) supplemented with protease and phosphatase inhibitor (Thermo Fisher Scientific). After the homogenate was centrifuged (4°C, 14,000 g, 10 min), the lower layer was extracted and boiled at 98°C for 5 min. Samples were electrophoresed in 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore). Primary antibodies were used to detect HSL, HSL-Ser563, and HSL-Ser660 (#4107, #4139, and #4126, Cell Signaling Technology, all by dilution of 1:2,000). Goat anti-rabbit IgG secondary antibody (GB23303, Servicebio, by dilution of 1:5,000) was used for luminescence. Actin (primary antibody No. AP0060, Bioworld Tech, by dilution of 1:5,000) was used as the loading control. The ECL (Solarbio) excited luminescence was collected and analyzed by the gel imaging system (Fusion Fx5-xt, VILBER LOURMAT). Overall results were represented by representative bands.