Protease inhibitor mix

To obtain the membrane proteins fraction, about 1 × 10⁸ trypomastigotes forms released from wild-type or LAMP-deficient fibroblasts, presenting about 70%–80% purity regarding amastigote forms contamination, were centrifuged during 10 min at 2,200g at 4°C and washed two times with PBS (pH 7.2). Samples were resuspended in 0.1M sodium carbonate lysis buffer (pH 11.5) supplemented with protease inhibitor cocktail (Protease Inhibitor Mix, GE Healthcare, USA), submitted to two ultrasonic pulses during 20 s on ice and incubated for 1 h on ice before ultracentrifugation at 150,000g for 90 min at 4°C (Sorvall Ultra Pro 80 Ultracentrifuge). Pellets were washed one time with 500 mM triethylammonium bicarbonate (TEAB) and one time with 50 mM TEAB.
Samples were suspended in 6M urea, 2M thiourea, and 50 mM TEAB buffer and reduced with 10 mM dithiothreitol (DTT) at (RT) for 1 h. After reduction, free thiols were alkylated with 20 mM iodoacetamide (IAA) in the dark for 1 h (RT). Samples were digested for 3 h (RT) in 1:100 ratio with Lys-C. Afterwards, samples were diluted eightfold with 50 mM TEAB (pH 8.0) and proteins digested with 1:100 ratio trypsin (Promega, Madison, WI) for overnight at 37°C. The reaction was quenched by adding formic acid (FA; 1% final concentration, Sigma-Aldrich, St. Louis, MO) followed by centrifugation at 14,000g for 10 min.

B. schlosseri haemocytes, collected as described above, were incubated for 30 min in FSW in the presence or in the absence (control) of 2 × 10⁹B. clausii spores (Enterogermina, Sanofi, Opella Healthcare Italy S.r.l., Origgio, Italy). They were then centrifuged at 2000× g for 5 min, the supernatant was collected, and 1% (vol:vol) of Protease Inhibitor Mix (GE Healthcare Europe GmbH, Freiburg, Germany) was added before its storage at −20 °C until use as CM in microbial cell mortality assay, as described above.

Total cell lysates were prepared by lysing B35 or C6 cells in mammalian protein extraction buffer (GE Healthcare Bio-Science, Uppsala, Sweden) supplemented with Protease Inhibitor Mix (GE Healthcare Bio-Science). To examine the protein expression levels, 5–80 μg of total protein extract of B35 or C6 was analyzed by separating in 10-15% sodium dodecyl sulfate polyacrylamide gels accordingly. The polyvinylidene difluoride membranes were used for transferring separated proteins. The membrane was then blocked with Membrane Blocking Solution (Life Technology, Frederick, MD, USA) for 1 h. The blocked membranes were then incubated with specific primary antibodies at 4°C for 12 h, followed by incubation with the respective horseradish peroxidase-conjugated secondary antibodies at room temperature for 4 h. Amersham ECL kit (Amersham, Bucks, UK) was used to develop and to reveal the signals of protein bands.

Protein extraction was performed as previously described with minor modifications^{8 (link)}. Briefly, four biological replicates, each consisting of a pool of 20 virgin females, were used for both phenotypes (CO vs. CA). After grinding to fine powder in liquid nitrogen and precipitation with 10% trichloroacetic acid in acetone for 2 h at −20 °C, samples were lysed in 30 mM Tris buffer pH 7.4 containing 8 M urea, 4% CHAPS, protease inhibitors (Protease Inhibitor Mix, GE Healthcare, Vélizy Villacoublay, France), and phosphatase inhibitors (Halt^TM Phosphatase Inhibitor Cocktail, ThermoFisher Scientific, Illkirch, France), using an ultrasonic processor (Bioblock Scientific, Illkirch, France) as previously described^{8 (link)}. After centrifugation (16,000 g for 20 min at 4 °C) to remove cellular debris and ultracentrifugation at 105,000 g for 1 h at 4 °C, the cytosoluble proteins were stored at −80 °C until analysis, and total protein concentration in each sample was determined using the Bradford Protein Assay Kit (Biorad, Marnes-la-Coquette, France) according to the manufacturer’s instructions.

Bacterial cell pellets were resuspended in 50 μL lysozyme and 100 μL 100 mМ TrisHСl pH 7.6, with 3 μL of Protease inhibitor Mix (GE Healthcare, USA). Cells were disrupted using a bead-beating homogenizer (MPBio, FastPrep-24, USA) with 0.5 mm silica-zirconium beads for 4 min, followed by 5 min on ice. For protein solubilization, SDS (Panreac, Spain) was added to the collected suspension to a final concentration of 10%. To reduce disulfide bonds, DTT (BioRad, USA) was added to a final concentration of 100 mM. Samples were then incubated at 60 °C for 30 min, centrifuged at 13,000 g at 4 °С for 5 min, and the supernatant used as protein solution. Protein concentration was measured by the Bradford method using the Bradford Protein Assay Kit (Bio Rad, USA).

Rho protein activity was analyzed using the RhoA/Rac1/Cdc42 activity assay kit (Cell Biolab, San Diego, CA, USA). In brief, cells cultured in a 10 cm dish were washed with PBS and lysed in 0.5 mL of lysis buffer containing protease inhibitor mix (GE Healthcare Bio-Sciences) and phosphatase inhibitors (2 mm NaF and 1 mm Na₃VO₄). After centrifugation at 14,000 × g for 5 min, 300 μg of protein from each cellular lysate was adjusted to a total volume of 1 ml with 1 × assay lysis buffer. Then, each sample received 40 μl of resuspended Rhotekin RBD or PAK PBD agarose beads, and all assay mixtures were incubated for one hour at 4°C with gentle shaking. The beads were then collected by centrifugation at 14,000 × g for 10 s followed by removing the supernatants and washing the bead pellets three times with 0.5 ml of 1 × assay lysis buffer. The beads were resuspended in 40 μl of 2 × SDS-PAGE sample buffer and boiled for 5 min. The samples were then analyzed by western blot and detected with anti-RhoA, anti-CDC42, or anti-Rac1 antibodies.

For analysis of TTR expression levels six heads from1-day old flies, per genotype, were homogenized in 20 μL of Laemmli Sample Buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 100 mM β-mercaptoethanol, 10% glycerol and 0.1% bromophenol blue) and snap frozen. Prior to SDS-PAGE analysis samples were heated at 100 °C for 5 min and centrifuged at 13,000 rpm for 5 min.
For analysis of soluble and insoluble fractions thirty adult fly heads, and fifty third instar larval brain and imaginal discs, were used per genotype. Tissue homogenization was performed in 100 μL of extraction buffer (PBS with 1% triton X-100 (Sigma-Aldrich), protease inhibitors (Protease Inhibitor Mix GE Healthcare, 80-6501-23, 1:100) and sodium orthovanadate 1 mM) for 30 s. Extracts were centrifuged at 15,000g for 10 min at 4 °C and supernatant was collected as the Triton-soluble fraction. The pellet was washed 3 times with extraction buffer and resuspended in 100 μL of 50 mM Tris pH 7.5 with 4% SDS and protease inhibitors followed by sonication. Extracts were centrifuged at 15,000g for 10 min at 4 °C and supernatant was collected as the Triton-insoluble fraction. SDS-PAGE and western blot analysis were performed as described below.