Mouse anti pgk1

Western blot analysis was used to investigate the protein content for Pgm2p-GFP fusion protein. Different strains were grown in media treated with and without LiCl. Protein extraction was performed as described by Szymanski [26 ]. Bicinchoninic acid assay (BCA) was performed to estimate protein concentration as described by the manufacturer (Thermo Fisher^®). 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 (Santa Cruz^®) was used to detect protein levels of Pgm2p-GFP. Mouse anti-Pgk1 (Santa Cruz^®) was used to detect Pgk1 protein levels used as internal controls. 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^®). Experiments were repeated at least three times; t-test analysis (P-value ≤ 0.05) was used to determine statistically significant results.

RNA immunoprecipitation assays were performed using Magna RNA‐binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions. Cells were homogenized in100 μl RIP lysis buffer with 0.5 μl Protease inhibitor and 0.25 μl RNase inhibitor on ice for 5 min and then stored at −80°C. Magnetic beads were incubated with 5 μg Mouse anti‐PGK1 (Santa Cruz Biotechnology, sc‐130335) and 5 μg Mouse anti‐lgG (Millipore, CS200621) at room temperature for 30 min, respectively. RIP lysate and beads‐antibody complex were incubated with RIP immunoprecipitation buffer containing 35 μl of 0.5 M EDTA, 5 μl RNAse inhibitor and 860 μl RIP wash buffer at 4°C overnight. Co‐precipitated samples were digested by proteinase K buffer containing 117 μl RIP wash buffer, 15 μl of 10% SDS, 18 μl of 10 mg/ml proteinase K at 55°C for 30 min with shaking and then the immunoprecipitated RNAs were extracted and purified for utilizing. The enrichment was determined by qPCR and RT‐PCR with agarose gel electrophoresis analysis.