Figure 2. Size fractionation and trypsin digestion of HBEC conditioned medium still resulted in RV-induced β₂ adrenoceptor desensitization on ASMCs.

HBEC (n = 6) were uninfected (Control) or infected with replication competent RV (RV) at an MOI = 2 for 24 hours to generate conditioned medium. The conditioned medium was pooled and serially fractionated through ultrafilters with molecular weight cut offs ranging from 100-3 kDa. Alternatively, conditioned medium from HBEC (n = 3) that was uninfected (Control) or exposed to: UV inactivated RV (UVi-RV) or replication competent RV (RV) at an MOI = 2 for 24 hours was digested in the presence of 500 µg/mL of trypsin for 24 hours at 37°C and trypsin removed using a 3 kDa MWCO ultrafilter. ASMCs (n = 6) were then treated with unfiltered or size fractionated control or RV-induced conditioned medium (A) or 3 kDa fractionated conditioned medium which was undigested or digested with trypsin (B) for 3 days. Isoprenaline induced cAMP was measured using a cAMP functional assay. Data represent mean ± SEM. Statistical differences were detected using multiple paired Student’s t-tests (A) or a 1-way ANOVA with Bonferroni post test (B) comparisons to control conditioned medium *p<0.05.