Trans blot semi dry transfer

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.

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.

To knock down TRMT6/61A in cell lines, 10 nM final total concentration of siRNA (Dharmacon ON-TARGETplus, TRMT6: #L-017324-02-0005, TRMT61A: #L-015870-01-0005) was transfected twice with Lipofectamine RNAiMax (Thermo Fisher #13778150) and collected 96 h post first transfection (48 h post second transfection). Non-targeting control siRNA (Dharmacon #D-001810-10-20) was used as negative control. Knock-down efficiency was confirmed by both RT-qPCR (Fig. 3d and Supplementary Fig. 3b, primer sequence in Supplementary Table 7) and western blot (Fig. 3d and Supplementary Fig. 3a) by TRMT6 mouse monoclonal antibody (SCBT #sc-271752, used at 1:1000 dilution). β-actin was probed by mouse monoclonal antibody (SCBT #sc-47778, used at 1:2000 dilution) as loading control. Western blots were performed with whole cell lysates on 12% SDS-PAGE and transferred to 0.45 μm nitrocellulose membrane (Whatman Protran BA85) by Trans-Blot Semi-Dry Transfer (Bio-Rad). Blocking and antibody incubation were in 3% milk in PBS with 0.05% Tween-20.