Figure 6.

Reduction of the amount of signal-transducible type I CD40 by expression of type II isoform. (A) mRNA levels of CD40 type I and II were analyzed by RT-PCR using primers P1 and P7 (Fig. 1A) and cytoplasmic RNAs from control cells (Con; transfected by using vector only), E-I/Con, E-I/II-1, and E-I/II-2. These transfectants were generated by using E-I (Fig. 5A) as a parent and the type II expression plasmids or the vector only. Low level type II mRNA expression was shown by using a longer-exposed film (L). (B) RT-PCR shown in A was analyzed also by using a PhosphorImager. Intensity of CD40 type I and II bands were normalized to HPRT levels and compared with that of type I in E-I (100%). (C) Proteins (5 μg) in membrane-rich fraction from nonactivated (Non) and LPS-activated (LPS) indicated cells were analyzed by immunoblotting using T-20 antibody (type I-specific). The intensities of these bands were compared with those of type I in nonactivated or LPS-activated E-I cells (shown below the blots). (D) In vitro translation was performed by using [³⁵S]Cys and 5 μg poly(A)⁺ RNA from the indicated cells or in vitro-synthesized CD40 RNA from Sp6 promoter (CD40 RNA). Immunoprecipitation was performed by using type I-specific T-20 antibody. Type I is indicated by an arrow. (E) Type I protein was detected in total-cell lysates (Total) and culture supernatants (Sup) from indicated cells by immunoblotting using T-20 antibody. The 45- and 27-kDa molecules were detected in type I transfectants. The 27-kDa molecules in total lysates from indicated cells (27 kDa) were detected by immunoblotting using T-20.