Phosphatase inhibitor cocktail 2 and cocktail 3

In 48 well plates, murine macrophage J774.A1 or RAW264.7 cells were co-incubated with assorted pharmacological compounds, including CC, AICAR or berberine for 3 hrs. The media containing the compounds was then removed. For Western blots, the compound-treated cells were washed 3 times with PBS, pH7.4, and lysed with RIPA buffer (G Biosciences), supplemented with a SigmaFAST protease inhibitor tablet (Sigma Chemical, Inc.) and phosphatase inhibitor cocktail 2 and cocktail 3 (Sigma Chemical, Inc.), and then harvested. The protein extracts were boiled for 5 min in sample buffer (Thermo Scientific, Inc.) containing 200 μM DTT, separated by SDS-PAGE, transferred onto PVDF membrane and blotted with the indicated antibodies. The blots were detected using an enhanced chemiluminescence kit (Pierce, Inc.). Blots were stripped and re-probed for assessment of protein levels using GAPDH (Glyceraldehyde 3-phosphage dehydrogenase) as a loading control. Densitometry of the blots was performed using ImageJ (http://rsbweb.nih.gov/ij/). For CFU assays, the treated cells were washed with fresh drug-free medium 3 times, and then infected with C. neoformans as described above. To test the effects of the examined chemicals on the activity or expression of fungal virulence determinants, we measured urease activity and capsule as described (Feder et al., 2015 (link); Kwon-Chung et al., 1987 (link)).