Nlrx1

Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 1 h before lysis with non-denaturing lysis buffer [20 mM Tris HCl pH 8, 137 mM NaCl, 1% Nonidet P-40 (NP-40), 2 mM EDTA] supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 25 mM N-ethylmaleimide (NEM). Whole cell lysates were incubated with antibodies against NLRX1 (Cell signaling), TUFM (Abcam), or rabbit IgG isotype control (GeneTex) at 4°C overnight followed by mixing with Protein A agarose beads (Merck Millipore) at 4°C for another 4 h. Lysate beads mixture was washed with IP washing buffer (0.1% NP-40 in PBS). The immunoprecipitates were eluted by being boiled in PhosphoSafe Extraction Reagent cell lysis buffer (Novagen) containing 1 × SDS-PAGE Loading Buffer (BIOTOOLS) and subjected to Western blotting.

Ginsenoside F₂ was isolated previously from leaves of Panax ginseng by a series of chromatographic procedures [9 (link)]. Ginsenoside F₂ has a molecular mass of 784 Da and was isolated with 98% purity. Primary antibodies of PRR5, CISD2, Bcl-2L, NLRX1, RPS15, RPL26, p53, PUMA, Beclin-1, UVRAG, AMBRA-1, mTOR, LC3-II, LC3-I, and β-actin together with all secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Atg5, Atg7, and Atg10 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).