Figure 5.

Detection of transferrin and its mRNA from cell culture. (A) Northern blotting. Total RNA was isolated from C7-10 cells (lanes 1, 2, 5, and 6) and Aag2 cells (lanes 3, 4, 7, and 8) by using Qiagen RNeasy mini kit (Qiagen, Chatsworth, CA), and 10-μg samples were electrophoresed on a 1% agarose gel containing formaldehyde. Lanes 1–4 show hybridization signal with A. aegypti transferrin cDNA labeled with [α-³²P]dATP to a specific activity of 10¹⁰ cpm/μg. Lanes: 1 and 3, control cells; 2 and 4, induced cells (16 h in E-O medium); 5–8, ethidium bromide-stained gel. (B) Western blotting. Antiserum against recombinant transferrin was used as the primary antibody to detect the mosquito transferrin in cell culture supernatants from 1 × 10⁷ C7-10 cells (lanes 1 and 2) and 3 × 10⁶ Aag2 cells (lanes 3–6). Lanes 1, 3, and 5, supernatant from control cells; lanes 2, 4, and 6, supernatant from treated cells used for the RNA samples shown in A. Lanes 1–4 were treated with a 1:5,000 dilution of immune serum; minor bands of lower molecular mass may represent degradation products. In lanes 5 and 6, preimmune serum was used in place of primary antibody.