Rabbit anti fak

Several antibodies directed against the extracellular part ADAM15 were used: a goat polyclonal (# AF935) and a mouse monoclonal (# mab945) from R&D Systems. Rabbit anti-ADAM15 (cytoplasmic domain, # ab84834), rabbit anti-PABP (# ab21060), rabbit anti-tubulin (# ab134185) and rabbit anti-calnexin antibody (# ab22595) from Abcam. Mouse anti-CD25 (# 174–820) from Ancell-Enzo Life Sciences GmbH. Mouse anti-puromycin (# MABE343, clone 12D10), mouse anti-myc antibody, (# 05–724), rabbit anti-alpha5 integrin (# AB1921) from Merck Millipore. Rabbit anti-FAK (# AHO0502) was from Invitrogen. Anti-Glutathion-S-Transferase peroxidase conjugate (# A7340) from Sigma-Aldrich.

The following antibodies were used: rabbit antibodies against WAVE2 (1:1,000, Cell Signaling Technology, catalog no. 3659), Integrin β1/ CD29 (GeneTex, catalog no. GTX128839), and DGCR8 (Abcam, catalog no. Ab191875); mouse antibodies against β-actin (1:5,000, Sigma), WAVE1 (1:200, Santa Cruz Biotechnology, catalog no. Sc-136120), and WAVE2 (1:1,000, Santa Cruz Biotechnology, catalog no. Sc-373889). Rabbit anti FAK (1:1,000, Invitrogen, catalog no. UB281522), rabbit anti-phospho-FAK (1:1,000, Abcam, catalog no. ab81298), rabbit anti Src (1:1,000, Cell Signaling Technology, catalog no. 2109S), rabbit anti phospho-Src (1:1,000, Cell Signaling Technology, catalog no. 6943S), goat horseradish peroxidase–conjugated anti-mouse IgG and goat horseradish peroxidase–conjugated anti-rabbit IgG were from Bio-Rad (1:2,000). ECL reagent was from Thermo Fisher Scientific. For immunoblotting, primary antibodies were made in 5% BSA and secondary antibodies were made in 5% non-fat dry milk (NFDM).

hDCs infected or not with L. amazonensis, L. braziliensis or L. infantum were fixed with 4% (v/v) paraformaldehyde for 15 min, washed with PBS, and submitted to cytospin centrifugation. Coverslips were quenched with 15 mM NH₄Cl for 20 min and washed three times with PBS, incubated in a blocking solution (3% (v/v) bovine serum albumin (BSA) in PBS) for 1 h, washed three times with PBS, permeabilized with 0.15% (v/v) saponin–PBS (Sigma- Aldrich, Saint Louis, MO, USA) for 15 min and then incubated with 1:500 rabbit anti-FAK (0.5 µg/mL) (Invitrogen, catalog number RC222574) or 1:100 rabbit anti-paxillin (0.1 µg/mL) (Invitrogen, catalog number QF221230) diluted in 1% (v/v) PBS + 0.15% (v/v) BSA saponin for 1 h. Next, anti-rabbit Alexa fluor 594 (Molecular Probes, catalog number A1011) was added and incubated for 1 h. Cells were then mounted with Prolong Gold antifade reagent and DAPI for nuclear staining (Invitrogen, Carlsbad, CA, USA). Images were acquired on a Leica confocal microscope using a 63×/1.4 objective and analyzed using Fiji Image J software.