4 to 20 tris glycine gradient gels

In vitro internalization assays with wild-type PA and other PA mutants proteins were performed at 37°C using DC 2.4 cells. DC 2.4 cells were grown in six-well plates to 90% confluence. DC 2.4 cells were incubated with PA proteins for 90 min at 37°C. For internalization inhibition studies, DC 2.4 cells were pretreated either with chlorpromazine (50 µM) or rottlerin (5 µM) for 30 min before the addition of PA proteins. After pretreatment, PA proteins were added, and further incubation was continued at 37°C for 90 min in the presence of inhibitors. After incubation, DC 2.4 cells were washed three times with phosphate-buffered saline (PBS), treated for 5 min with low-pH buffer (50 mM glycine and 100 mM NaCl, pH 4.0) to remove the PA proteins bound to the cell surface and again washed three times with PBS. The DC 2.4 cells were lysed with mammalian protein extraction reagent (MPER) lysis buffer (Pierce) containing 1× Halt protease inhibitor cocktail and 10 U of DNase/ml. The cell lysates (30 µg of total protein) were subjected to SDS-PAGE analysis using 4 to 20% Tris-glycine gradient gels (Novex, San Diego). Proteins were then transferred to nitrocellulose membranes, followed by Western blotting using anti-PA antibodies.