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| Protein Electrophoresis and Western | Pellets were then resuspended in 1× SDS–PAGE sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 1% β-mercaptoethanol, 6% glycerol, 0.004% bromophenol blue) for western blot analysis. Proteins were resolved using SurePAGE gels (GenScript, M00653) with MES running buffer (GenScript, M00677) and transferred onto 0.2 μm nitrocellulose membranes (LI-COR, 926-31092). Membranes were blocked for 1 h at room temperature in 5% milk prepared in 1× PBST (0.1% Tween-20). After blocking, antibodies diluted in 2% milk in 1× PBST were incubated with membranes. Primary antibodies used: Flag (Genscript, A00187) and GAPDH (Proteintech, 60004-1-Ig). Samples were resolved on a SurePAGE gel (Genscript, M00653) using MES buffer (Genscript, M00677) and transferred onto a 0.2 μm nitrocellulose membrane (LI-COR, 926-31092). Membrane was blocked in 5% milk in 1× PBST (0.1% Tween-20) for 1 h at room temperature. After blocking, antibodies diluted in 2% milk in 1× PBST were incubated with membranes. Primary antibodies used: 6× His (Genscript, A00186), RPS6 (Cell Signaling, 2217), RPL4 (Invitrogen, cat. no MA5-56865). | Get A Quote |
Cellular dormancy enables survival during prolonged nutrient limitation by reversibly suppressing protein synthesis1,2,3,4. How inactive eukaryotic ribosomes are reactivated when nutrients return remains unclear. Here, using high-resolution in situ cryo-electron tomography in Schizosaccharomyces pombe, we identify SNOR, an SBDS domain-containing ribosome-associated factor that binds at the peptidyl transferase centre and contacts the hypusinated loop of eIF5A during glucose depletion-induced dormancy. Rather than acting as a canonical hibernation factor, SNOR licenses dormant ribosomes for rapid translational restart. Upon glucose repletion, SNOR and eIF5A act together to promote efficient recovery of polysomes... More
