Figure 5.

The CAST^(1–68) polypeptide impairs protein synthesis and leads to accumulation on polysomes following proteasome inhibition. (A) A graphical illustration of the Gimap3 and YFP constructs. AIG, Gimap family domain; CC, coiled coil; TM, transmembrane domain. (B) Immunoblotting with the CAST polyclonal antibody comparing immunoreactivity of HEK293 lysates generated with different buffers and detergent combinations. The standard lysis buffer is used in Figure 4, whereas the ribosome lysis buffer is optimized for polysome profiles. (C and D) Polysome profiles of YFP and CAST^(1–68)–YFP. Total cell lysates from HEK293 cells transfected with the indicated cDNAs were prepared with the ribosome lysis buffer and separated on 10–50% sucrose gradients. SM, starting material. Top: Absorbance at 254 nm of 82 fractions collected from the top. Below, immunoblotting of TCA precipitated fractions from five pooled aliquots from the top with the indicated antibodies and with equal film exposures ±MG132