Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

A total of 100 mg GST or GST-RAB5 purified and bound to nickel-agarose resin (Sigma, P6611) was used to affinity isolate 100 mg of lysate from E. chaffeensis-infected THP-1 cells (lysis buffer: 43 mM Na 2 HPO 4 , 14.7 mM KH 2 PO 4 , 137 mM NaCl, 27 mM KCl, pH 7.3, 1% [v/v] Triton X-100 [Sigma, T8787] and 1% protease inhibitor cocktail III) that were precleared with nickel-agarose resin. Western blotting with rabbit anti-Etf-1 was carried out as described. 43 Affinity isolation using rEtf-1 (C-terminal 250 residues) containing an N-terminal 6£His-tag 43 was performed as described with minor modifications. 131 Purified and nickel-agarose-bound rEtf-1 (0.5 mg) was added to lysates from 3 £ 10 7 uninfected THP-1 cells precleared with nickel-agarose. After extensive washing with phosphate buffer (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl) with 1% (v/v) NP40, the bound proteins were eluted with phosphate buffer containing 250 mM imidazole and precipitated with 10% trichloroacetic acid. After centrifugation, the pellet was neutralized with 1.5 M Tris-HCl (pH 8.8) and dissolved in 2£ SDS-PAGE loading buffer. Western blotting was performed with rabbit anti-RAB5, rabbit anti-PIK3C3, and mouse anti-BECN1.