Fig. 1.

Transient expression of eIF3i in CHOK1 cells is limited in extent and with little effect on global protein synthesis levels. A. Immunoblot detection of eIF3i in CHOK1 and HEK cell lysates prepared from cells transiently transfected with eIF3i in pcDNA3.1V5 with (3iV5) or without (3i) read-through to the V5 tag (v, vector control) and maintained at 37 °C for 24 h. B. Immunoblots generated from 3 independent experiments of the type shown in Fig. 1A were quantified with NIH ImageJ. (V) is the upper band of the 3iV5 samples in (A) and (i) the lower band of the 3iV5 samples in (A) whilst V + I is the sum of these two bands in the immunoblots of V5-tagged 3i transfections. Error bars represent ± 1SD. C. Co-expression of all 8 components of the molecular chaperone CCT with eIF3i did not improve the level of eIF3i expression. CHOK1 cells were co-transfected with 2 μg vector encoding human eIF3i and 1 μg each of vectors encoding each of the CHO cell CCT subunits with or without ³⁵S-labelling for 1 h at the end of the 24 or 48 h period. Western blots of cell lysates were probed for the indicated CCT subunits, eIF3i and tubulin as a loading marker while (D) ³⁵S-labelled samples were processed for scintillation counting, error bars represent ± 1 SD from 3 measurements.