Figure 3.

Western blot analysis of CD23 after stimulation of IL-4, GM-CSF, IL-4/GM-CSF. Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml) (vehicle control), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham). Paxillin mouse monoclonal IgG₁ (Transduction Laboratories) was used as an irrelevant isotype control.