The GAL::HA-efg1/ENP1-TAP strain was transformed with a pRS415-GPD plasmid expressing full length or truncated Efg1. Single clones of the transformants of similar size were cultured, 5-fold serially diluted and spotted onto plates containing SC medium with galactose or glucose. The plates were incubated at 30 or 37°C for 2 days or at 18°C for 4 days. Strains UTP9-TAP, efg1Δ/UTP9-TAP and efg1Δ/UTP9-TAP complemented with pRS415-GPD-EFG1 were spotted on YPD plates.
Immunoprecipitation was conducted as described below. For western blot analysis, proteins were separated in 4–20% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes (GE Healthcare) using a semi-dry electrophoretic transfer cell (BioRad). HRP-conjugated anti-Flag (1:10000, Sigma) and HRP-conjugated anti-HA antibodies (1:3000, CST) were used with appropriate dilution ratios. Ribosome profile assays were performed as previously described (21 (link)).
Immunoprecipitation was conducted as described below. For western blot analysis, proteins were separated in 4–20% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes (GE Healthcare) using a semi-dry electrophoretic transfer cell (BioRad). HRP-conjugated anti-Flag (1:10000, Sigma) and HRP-conjugated anti-HA antibodies (1:3000, CST) were used with appropriate dilution ratios. Ribosome profile assays were performed as previously described (21 (link)).
