Figure 2.

Analysis of Lec35 nucleic acids. Southern blot analysis of genomic DNA. ³²P probes were generated with specific PCR primers and covered either the complete coding sequence of SL15 cDNA (A), or exons 1 and 2, exon 3, exon X, or exons 4–7 (B). Genomic DNA (Easy-DNA; Invitrogen, San Diego, CA) from parental CHO-K1, Lec35.1, or Lec35.2 cells (10 μg for all probes except exon X [50 μg]) was digested with either BamHI or HindIII as indicated and analyzed by Southern blots according to standard methods (Sambrook et al., 1989). The positions of λ phage-HindIII markers are shown. In addition to a strongly hybridizing fragment of ∼6 kbp, a faint BamHI fragment of ∼10 kbp was reproducibly detected with the exon 3 probe in CHO-K1 DNA. Because exon 3 lacks a BamHI site, this is likely to be an irrelevant cross-reacting fragment. Northern blot analysis of RNA. Total RNA was isolated from the CHO-K1, Lec35.1, or Lec35.2 cells lines, as well as the 6C and 10A transfectants of Lec35.1 grown in the absence or presence of 10 ng/ml doxycyclin for 4 d, and 10 μg of total RNA was analyzed by gel electrophoresis and blotting (C and D) as described (Sambrook et al., 1989). A ³²P probe prepared from the complete coding sequence of SL15 cDNA was used. Relative intensities of the Lec35 mRNA bands, as described in the text, were measured with a phosphorimager (Fuji, Stamford, CT). The migrations of native (n) and recombinant (r) Lec35 transcripts (D) were reproducible. The position of a 1.35-kb RNA marker (Life Technologies) is shown (C).