Western Blotting of Cell Lysates for Protein Detection

Western blotting of whole cell lysates was performed as previously described (74 (link)). Briefly, normalized amounts of eluates were resolved by SDS-PAGE in 4-to-20% mini-Protean gels (Bio-Rad) at 110 V for 15 min, followed by 200 V for 25 min. Following transfer onto nitrocellulose membranes, blots were blocked and probed with 5% (vol/vol) nonfat dry milk in Tris-buffered saline plus 0.05% Tween 20 (TBS-T). Primary antibodies used were rabbit anti-ALIX (Abcam [ab88388]; 1:1000), rabbit anti-Hgs (Abcam [ab155539]; 1:1000), rabbit anti-Rab11a (Abcam [ab88388]; 1:1000), rabbit anti-Rab27a (Abcam [ab55667]; 1:1000), rabbit anti-Rab27b (Cell Signaling Technology [ab44813]; 1:1000), mouse anti-GAPDH (Santa Cruz [sc365062]; 1:1000), mouse anti-β-actin (Santa Cruz [sc47778]; 1:1000), or antisera targeting A. phagocytophilum P44 or APH1235. Rabbit P44 antiserum was generated by New England Peptide. Rabbit anti-APH1235 (45 (link)) was a gift from Dr. Erol Fikrig of Yale University, New Haven, CT. Secondary antibodies were horseradish peroxidase-conjugated horse anti-mouse IgG or anti-rabbit IgG (Cell Signaling Technology; 1:10,000). All blots were incubated with chemiluminescent substrates, imaged, and processed as described (74 (link)).