Figure 3.

Blastomyces dermatitidis and β-glucan induce mucin expression in the immortalized canine airway carcinoma (BACA) cells. BACA cells at 90% confluency were challenged with live (LV) or heat-killed (HK) B. dermatitidis strain SCB-2 at the indicated multiplicity of infection (MOI), or indicated concentrations of β-glucan for 24 hours. Control (Ctrl) cells were exposed to the same volume of sterile phosphate-buffered saline. Cytoplasmic and nuclear protein extracts were separated on acrylamide-agarose (mucins) and acrylamide [FOXA2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and H3] gels, respectively, and probed with specific antibodies against each protein. GAPDH and H3 were used as loading controls. The experiments were independently performed three times with similar results. Western blots from one typical experiment are shown. Additional replicates of Western blots can be found in Supplemental Figure S4. Expression levels of MUC5AC, MUC5B, and forkhead box protein A2 (FOXA2) were measured by ImageJ software version 1.52a. Densitometry data of FOXA2, MUC5AC, and MUC5B expression are expressed as means ± SEM from all three experiments (A and B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test).