Enhanced macrophage activation with higher concentration of immuno-purified hCLCA1.

(A) Representative silver stained SDS-PAGE gel showing immuno-purified hCLCA1 and a 2-fold dilution series of lysozyme. (B) Densitometry analysis using lysozyme standard curve demonstrated a higher concentration of pure hCLCA1 (9.425 ± 0.335 pg/μL) was immunoprecipitated with an optimized protocol. Result was presented as the mean of 3 samples ± SEM. (C) The mRNA expression of cytokines in macrophages stimulated with a higher concentration of hCLCA1 for 48 h was quantified using RT-qPCR. The fold difference was calculated against the corresponding control (immunoprecipitation of eGFP using hCLCA1-N14 antibody). Results were presented as the means of 4 samples ± SEM. (D) Representative Western blots showing intracellular IL-1β and GAPDH levels in immuno-purified eGFP or hCLCA1-stimulated macrophages. GAPDH was used as a loading control for densitometry analysis. Immuno-purified hCLCA1-stimulated macrophages had a 2.38 ± 0.21 folds increase in IL-1β protein levels over immuno-purified eGFP-stimulated macrophages (the hCLCA1-induced IL-1β was normalized to the eGFP-induced IL-1β in each sample). Results were presented as the means of 7 samples ± SEM. (E) Secreted cytokine protein expression in macrophages stimulated with a higher concentration of immuno-purified hCLCA1 was analyzed using Bio-plex Suspension Array System. The fold difference of each sample was compared against the corresponding control. Results were the means of 3 samples ± SEM. Significant fold differences from corresponding control values are indicated by * (p < 0.05), ** (p < 0.005) and *** (p < 0.001).