Tx 114

All solutions were prepared using double-distilled water. The active substance, FMT, was obtained from Sigma-Aldrich (USA). A standard solution of FMT (1687.40 μg mL⁻¹) was prepared by dissolving 0.169 g in 100 mL of water with the addition of a drop of concentrated hydrochloric acid.
The surfactants, SDS and TX-114, were obtained from Sigma-Aldrich. A standard solution of SDS (0.2 mol L⁻¹) was prepared by dissolving 5.768 g in a 100-mL volumetric flask with water. A stock solution of TX-114 (5% w/v) was prepared by diluting 100% (w/v) solution of TX-114 in a 100-mL flask.
The electrolytes, sodium chloride, calcium chloride, potassium bromide, and sodium sulfate, were obtained from Poch (Gliwice, Poland). Standard solutions of the chlorides (4 mol L⁻¹), bromide (4 mol L⁻¹) and sulfate (1.5 mol L⁻¹) were prepared by dissolving appropriate amounts in 100 mL of water.
Methanol, acetonitrile, dipotassium hydrogen phosphate, sodium hydroxide, and hydrochloric and phosphoric acids were obtained from Poch. A stock solution of hydrochloric acid and sodium hydroxide (0.1 mol L⁻¹) was prepared by dissolving appropriate amounts in a 1000 mL volumetric flask. Working solutions of hydrochloric acid and sodium hydroxide (10⁻² mol L⁻¹) were prepared by diluting the stock solutions (0.1 mol L⁻¹) in 100 mL of water.

M. bovis MAPs were fractionated using TX-114 as previously described [48 (link)] with minor modifications. In brief, M. bovis HB0801 pellet was re-suspended in PBS containing 4% TX-114 (Sigma) and 1 mM PMSF (Sigma) and kept for 3-5h at 4°C after scraping. Un-lysed cells and debris were precipitated and removed by centrifugation for 15 min at 15,400 g. The supernatant was then incubated for 20 min at 37°C and then centrifuged for 5 min at 7500 g to separate the two phases. The upper aqueous phase was discarded and the lower detergent phase was reconstituted to the original volume with 1 mM PMSF in PBS for washing. After washing, the proteins in the detergent phase were re-established in 100% methanol and incubated overnight at −80°C. After centrifugation for 20 min at 15,400 g, the proteins were re-suspended in lysis buffer. Protein concentration was measured with a 2D quant kit (GE Healthcare, Sweden).

Membrane-associated and ectodomain CD163 were separated using the Triton X-114 method (Tx-114, Sigma-Aldrich, Brøndby, Denmark)40 (link). In short, plasma and cell lysate were precleared to remove cellular debris using differential centrifugation step (30 min at 2000 g followed by 45 min centrifugation of resulting supernatant at 12000 g). Subsequently samples were diluted 1:10 in a precondensed 2% Tx-114 solution in 1x PBS pH 7.4 and incubated for 10 min on ice. Samples were then heated to 37 °C for 10 min in order to induce separation of the aqueous and the detergent phase after which samples were centrifuged at 22,500 g for 10 min at 37 °C. The aqueous phase were then harvest and kept at 4 °C for later analysis. The detergent phase were further washed twice with 0.1% Tx114 after which protein was harvested for analysis by chloroform:methanol (ratio 4:1) precipitation. Precipitated protein was subsequently resuspended in 1x LDS sample buffer (Thermo Fischer Scientific, Slangerup, Denmark) by boiling.

CIPRO, LEVO, TX-114, triethylamine (TEA), sodium monophosphate, and orthophosphoric acid were purchased by Sigma Aldrich (Steiheim, Germany). MOXI was purchased by Santa Cruz Biotechnology (Dallas, TX, USA). Acetonitrile (ACN), methanol (MeOH), and potassium chloride were purchased by Merck (Darmstadt, Germany). All reagents were HPLC-grade. Sodium chloride, sodium hydroxide, boric acid, and acetic acid were purchased by Avantor Performance Materials (Gliwice, Poland). The HPLC analysis was done on LiChroCART^® 250-4, HPLC-Cartridge, and LiChrospher^® 100 RP-18 (5 μm) (Merck, Darmstadt, Germany) with a LiChroCART^® guard column (4-4, LiChrospher^® 100 RP-18 (5 μm)).