Complete edta free anti protease cocktail

Ehrlichia ruminantium Gardel virulent strain (passages 17, 19, and 24) and ERGatt (passages 240, 242, and 244) variants were grown in bovine aortic endothelial cells (BAECs), as described elsewhere (Marcelino et al., 2015 (link)). When 80% cell lysis was observed, infectious and extracellular EBs were harvested and then used to (i) infect a new confluent monolayer of BAEC or (ii) to be purified using a multistep centrifugation methodology (Peixoto et al., 2007 (link)). Purified EBs were stored at -80°C with a “Complete EDTA-free” anti-protease cocktail (Roche, Germany) and anti-phosphatases (Roche, Germany) prior to proteomic analysis.

Infected blood from ERG-infected goats was loaded on finite cultures of bovine aortic endothelial cells (BAE) to isolate the ERGardel virulent strain (ERGvir). ERGvir was then routinely propagated in BAE as described elsewhere [36 (link),37 (link)]; ERGvir samples from passages up to passage 44 were used throughout this study, as they have been proven to be highly infectious in previous studies [38 (link)]. In order to obtain an attenuated ERG strain, ERGvir was cultivated over 230 passages in BAE cells, as suggested by Martinez (1987)[14 ]. When 80% cell lysis was observed (at 120hpi for ERGvir and 96hpi for ERGatt, S1 Fig), supernatant and cellular debris containing infectious ER elementary bodies were harvested and then used to (i) infect a freshly confluent monolayer or (ii) to be purified using a multistep centrifugation methodology [39 (link)]. Purified ERs were stored in SPG [39 (link)] at −80°C with a “Complete EDTA-free” anti-protease cocktail (Roche, Germany) prior to proteomic analysis. Prior to in vivo assays, purified ER were stored in SPG in liquid nitrogen [38 (link)].
Growth of ERGvir and ERGatt was monitored by phase-contrast light microscopy and quantified as previously described [17 (link)].

GST pull-down assays were prepared as previously described^{47 (link)}. Briefly, 2 μg of recombinant His₆-pTKL_RVxF1, pTKL_KD_WT or pTKL_KD_KRIMAS was incubated with PfPP1c-GST, PfeIF2γ-GST (negative interaction control) or GST bound to glutathione-Sepharose beads, and 25 µg of bovine serum albumin (BSA) in binding buffer (20 mM Tris-HCl pH 7.4, 500 mM NaCl, 20 mM HEPES, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM DTT, Roche cOmplete EDTA-free anti-protease cocktail, 1 mM MnCl₂, 50 µM ZnCl₂) for 2 h at 4 °C on a rotating wheel. After three washes, proteins were analyzed by western blot using an anti-His₆ antibody (diluted 1:1000) or an anti-GST antibody (diluted 1:2000).