Anti phosphotyrosine

Tissues were homogenized in hypotonic buffer with Triton X-100 while using metal beads in a Tissue Lyser (Qiagen, Hilden, Germany) as previously described [19 (link)]. 40–50 μg of total protein was resolved per lane by SDS-PAGE and transferred to a nitrocellulose membrane. Blots were probed with specific antibodies against GRK2 (sc-562, Santa Cruz Biotechnology, Dallas, TX, USA), β-Arrestin 1 and 2 [20 (link)], anti-pAkt (Ser473 #9271, Cell Signalling, Danvers, MA, USA), total Akt (#9272 Cell Signalling), β-Actin (127M4866V, Sigma, San Luis, MO, USA), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, sc-32233, Santa Cruz), anti-phospho-tyrosine (#61-5800, Invitrogen, Carlsbad, CA, USA), anti-insulin receptor β subunit (sc-57342, Santa Cruz), anti IGF1R (#9750, Cell Signalling), and α-Tubulin (sc-53030, Santa Cruz). Immunoreactive bands were visualized while using enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) or the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). Films were scanned with a GS-700 Imaging Densitometer and then analyzed with Quantity One Software (Bio-Rad, Hercules, CA, USA), or using an Odyssey Classic reader and the Odyssey software package 3.0 (Li-Cor Biosciences).

Cells were cultured in 10 cm dishes, irradiated at 2 Gy and lysed after 5 min using lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% Na Deoxycholate, supplemented with complete proteases and phosphatases inhibitors (Roche, Boulogne-Billancourt, France )). Further, 2 mg of protein lysate was pre-cleared with 30 µL agarose protein G beads (1:2 diluted in lysis buffer) by incubating for 3 h at +4 °C on an orbital shaker. After, the cleared sample was transferred to a microcentrifuge tube containing 5 µg of IgG2b isotype control (MAB004—R&D Systems) or 2.5 µg anti-phosphotyrosine (03–7700- Invitrogen, ThermoFisher Scientific, Villebon-sur-Yvette, France) and 2.5 µg anti-phosphotyrosine (05–321-Merck—Millipore, Guyancourt, France) antibodies and left to incubate overnight at +4 °C on an orbital shaker. The following day, 30 µL agarose protein G beads (1:2 diluted in lysis buffer) were added to the sample and incubated for 1 h at +4 °C on an orbital shaker. After 5 washes with lysis buffer, the supernatant was aspirated and 25 µL of warm loading buffer was added to the beads, vortexed and boiled for 10 min. 15 µL of sample was loaded in SDS-PAGE gel and electrophoresed. The sample was transferred to a nitrocellulose membrane and probed with TYRO3 antibody.

Trypomastigotes (ECM-treated for 120 min or control) were fixed in 2% paraformaldehyde for 15 minutes at room temperature, pelleted by centrifugation (4,000 x g for 5 minutes), washed twice in PBS, resuspended in PBS, added to a coverslip and dried at room temperature. After permeabilization of the parasites with PBS containing 1% BSA and 0.1% Triton X-100 for one hour at 37°C, anti-phosphoserine, anti-phosphothreonine, anti-phosphotyrosine (Invitrogen–dilution 1:200 for each antibody), anti-PAR monoclonal antibody (1:200) or anti–TcHexokinase (kindly provided by Dr. Ana Cáceres, Universidad de Los Andes, Venezuela) were added and incubated for 1 h at room temperature. After three washes with PBS containing 0.1% Triton X-100, the correspondent secondary antibodies were added (anti-rabbit or anti-mouse-Alexa 555 conjugated (1: 5000); followed by one hour incubation at 37°C. After three washes in PBS-0.1%-Triton X-100, the coverslips were faced under a solution containing 50% glycerol, 50% milliQ H₂O 2 mM sodium azide, and 20 μg/mL of 4',6-diamidino-2-phenylindole, dilactate (DAPI-Invitrogen). The images were taken on an ExiBlue™ camera (Qimaging®) coupled to a Nikon Eclipse E 600 optical microscope and deconvoluted using the software Huygens Essential (Scientific Volume Imaging).