Figure 4.

The N terminus of CAST Gimap3 impairs protein synthesis. (A) A graphical illustration of the Gimap3 constructs used. AIG, Gimap family domain; CC, coiled coil; TM, transmembrane domain. (B) SDS–PAGE of in vitro synthesis of proteins from a T7 promoter with [³⁵S]methionine and cysteine. Lanes numbered according to the constructs in A. (*) Consistent smearing of labeled protein. Arrow indicates labeled protein from CAST^(1–68)–YFP that comigrates with full-length YFP (lane V). Right: Four times excess sample was loaded for construct II. (C) Quantification of CAST^1–68–YFP in vitro production relative to YFP from six independent experiments. For the quantification, the smeared signal for CAST^(1–68)–YFP (* in B) was included along with the mature protein. (D) Quantification of CAST Gimap3 in vitro production relative to BALB Gimap3 from six independent experiments. (E and F) Immunoblotting of total cell lysates from HEK293 cells transfected with the indicated cDNA constructs and treated with the indicated antibodies. Cells were treated with or without MG132 7 hr prior to collection. (G) Immunoblotting of total cell lysates from HEK293 cells transfected with a Gimap3 cDNA construct and treated with the indicated antibodies. Cells were treated with or without MG132 7 hr prior to collection.