Rnase a from bovine pancreas

LAB were purchased from the Japan Collection of Microorganisms (JCM) or isolated from fermented foods (Table S1). Pediococcus acidilactici K15, Lactobacillus plantarum ATCC14197^T, and Lactobacillus pentosus ATCC8041^T were cultured at 30 °C for 24 h in MRS broth (Becton Dickinson, Sparks, MD, USA). Lactobacillus delbrueckii sup. bulgaricus ATCC11842^T and Lactobacillus rahmnosus ATCC53103^T (LGG) were cultured at 37 °C for 24 h in MRS broth. They were then heat-killed, washed twice with saline, and suspended in saline. For nuclease treatment of the heat-killed bacteria, treatment with RNase A from bovine pancreas (Sigma, St. Louis, MO, USA) was performed under low salt conditions (10 mM Tris-HCl, pH 8.0) or high salt conditions (10 mM Tris-HCl, 0.3 M NaCl, pH 8.0) at 37 °C for 2 h. RNase A-treated bacteria were washed twice with each buffer and used for subsequent experiments.

LPS was extracted by a combination of phenol water extraction as described previously (30 (link)) and purification using size exclusion chromatography to eliminate peptides, nucleotides, and other impurities. Briefly, bacterial cells were lysed in sodium dodecyl sulfate (SDS; Sigma-Aldrich) buffer, and proteins were removed by proteinase K digestion (Sigma-Aldrich). SDS was removed by ethanol precipitation (100% ethanol, ice-cold), and RNA and DNA were eliminated by RNase and DNase treatment (DNase I from bovine pancreas and RNase A from bovine pancreas; Sigma-Aldrich). Finally, the LPS was isolated by hot phenol-water extraction from the crude extract and further purified by size exclusion chromatography (Sepharose CL-6B column; Sigma-Aldrich) in the presence of SDS. The identity of LPS was confirmed by nuclear magnetic resonance (NMR; data not shown).

Hypotonic purification of mitochondria from ~5 x 10⁷ uninduced and tet-induced PF cmyc-Tb927.1.1730, cmyc-Tb927.10.1730, and cmyc-Tb927.10.7910 cells was carried out as described above. The lysis buffer was prepared either with 40 U of RNaseOUT (Invitrogen) or 200μg/ml of RNase A from bovine pancreas (Sigma). Anti c-myc agarose conjugated beads (Sigma) were washed five times with 1ml ice-cold PBS at 4°C and subsequently washed once with 1ml of ice-cold immunoprecipitation wash buffer (Tris-HCL, pH 8.0, 10mM, NaCl 100mM, NP-40 0.1%, 1X complete EDTA-free protease inhibitor (Roch) and 1% PBS). After the last wash, 50 μl of beads were re-suspended for each reaction in 1 ml of ice-cold wash buffer and incubated for 1 hour at 4°C on a tube rotator. After adding the mitochondrial lysate to the beads, mixture was rotated for 2 hours at 4°C followed by centrifugation at 500rpm for 1 min at 4°C. After removing the supernatant (unbound proteins), the beads were washed four times with 1ml of immunoprecipitation wash buffer and then resuspended in SDS-PAGE loading dye. Aliquots of lysate, bound and unbound fractions were loaded on 10% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membrane and probed with polyclonal antibodies against MRB 8170 and TbRGG2 (a generous gift from Laurie Read, state University of New York at Buffalo, USA).

Rosmarinic acid (95%) (Figure 1) was obtained from Extrasynthese (Genay, France) and dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany). Roswell Park Memorial Institute Medium (RPMI) 1640, F10, Phytohaemagglutinin (PHA), cytochalasin B, streptomycin, penicillin, phosphate buffered saline (PBS), methanol, heparin, sodium chloride, sodium bicarbonate, RNase A from bovine pancreas and propidium iodide 1.0 mg/mL solution were obtained from Sigma-Aldrich Chemicals S.A (Madrid, Spain). Fetal bovine serum (FBS) was obtained from Gibco (Life Technologies S.A., Madrid, Spain). Fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD71 transferrin receptor antibody was from Southern Biotech (Birmingham, USA).

U. maydis cells freshly cultured in YEPS medium at 2.0x10⁷ cells/ml were harvested by centrifugation at 2,500 rpm for 5 min at 30°C. The medium was discarded, and the cells were washed twice in 200 μl of TBS (50 mM Tris HCl pH 7.5, 150 mM NaCl), with centrifugation for 5 min at 2,500 rpm between washes. The cells were suspended in 100 μl of TBS and mixed thoroughly with 100 μl of fixative solution (50% v/v ethanol, 10% v/v CH₃COOH). The suspension was kept at RT for 5 min and centrifuged again to remove the fixative solution. Cells were washed twice with 200 μl of TBS, and the pellet was suspended in 100 μl of the same solution supplemented with 0.2 μg/ml RNase A from bovine pancreas (Sigma-Aldrich Co.). The cell suspension was kept at 4°C overnight, moved to 37°C for 1 h, centrifuged and washed once in 200 μl of TBS. The cell pellet was then suspended in 50 μl TBS with propidium iodide (2 μg/ml; Sigma-Aldrich Co.) and allow to absorb the dye for 6–8 h at 4°C. Stained cells were washed twice with 100 μl of TBS and resuspended in 50 μl of TBS. Then, calcofluor (Sigma-Aldrich Co.) was added at 25 ng/ml TBS, and after 5 minutes of treatment, the excess dye was removed by centrifugation and suspended in 25 μl of buffer for observation under a microscope.