Figure 5.

Function of CD40 isoforms. (A) RAW 264 stable transfectants were generated by using type I, II, III, and IV CD40 isoform expression plasmids carrying the cDNAs downstream of the elongation factor (EF)-1α promoter. Negative control cells were generated by transfection using vector only. Control (Con), type I (E-I), type II (E-II), type III (E-III), and type IV (E-IV) transfectants were activated by agonist CD40 antibody FGK-45 or LPS. IL-12 p40 and HPRT (as control) mRNAs were analyzed by RT-PCR using cytoplasmic RNAs from indicated transfectants. (B) Expression levels of endogenous CD40 mRNA were analyzed by RT-PCR using P1 and P2 (endogenous CD40-specific) primers (Fig. 1A) and cytoplasmic RNAs from nonactivated (N) and LPS-activated (L) control cells (Con) and type II (E-II) transfectants. (C) Expression levels of CD40, IL-12 p40, and HPRT mRNAs were analyzed by RT-PCR using cytoplasmic RNAs from negative control (Con), type I (C-I), and type II (E-II) transfectants and a type I+II (C-I/E-II) double transfectant. Expression of type I mRNA was under-controlled by the cytomegalovirus promoter in C-I and C-I/E-II, and expression of type II mRNA was under-controlled by the EF-1α promoter in E-II and C-1/E-II. Endogenous (Endo.) plus transfected (T) type I and endogenous type II mRNAs were amplified by using P1 and P2 primers (Fig. 1A). Transfected type II mRNA was amplified by using primers binding to 5′ and 3′ untranslated regions encoded in the expression vector. (D) Protein (100 μg) in membrane-rich fractions from nonactivated (N) and LPS-activated (L) control (Con) cells and C-I/E-II cells were analyzed by immunoblotting using L-17 (binding to N terminus of CD40) and T-20 (binding to C terminus of CD40, type I-specific) antibodies. (E) Nonactivated control cells (Con/Non, dotted line), LPS-activated control cells (Con/LPS, solid line), and LPS-activated C-I/E-II cells (C-I/E-II/LPS, filled with gray) were analyzed by flow cytometry using anti-CD40 antibody (FITC-conjugated 3/23, PharMingen).