FIG. 8.

The predominantly expressed protein in BCBL-1 cells is the 48-kDa product of reading frame 2. (A) (Top) Diagram indicating the structure of expression constructs (pBCBL-1 XhoII-NsiI-XprFr1, -XprFr2, and -XprFr3) which contain the Xpress epitope (Invitrogen) in different reading frames at the 3′ end. (B) (Left) Equivalent expression of the three constructs in transfected SLK cells was verified by Western blotting with MAb 5B2, the epitope for which is present in all three reading frames (see text). (Right) Western blotting of equivalent lysate loads was also performed with the anti-Xpress epitope MAb, demonstrating that translation of the Xpress epitope occurred predominantly in frame 2. Note that the frame 3 construct is overloaded to allow detection of the epitope tag. The tag is also detectable in frame 1 upon longer exposure (data not shown). (C) Western blotting of SLK cells transfected with pBCBL-1 XhoII-NheI (wild type [wt] [A, bottom]) demonstrates authentic translation of the predominant 48-kDa product as detected in BCBL-1 lysate (BCBL). Mutation of CUG (to UUG) in reading frame 2 at position 118679 in an equivalent transfected construct (mut) eliminates detection of the protein.