Puromycin

Leishmania mexicana (MNYC/BZ/62/M379) promastigotes were cultured at 25 °C in HOMEM medium (modified Eagle’s medium) supplemented with 10% (v/v) heat-inactivated foetal calf serum (GIBCO) and 1% Penicillin/Streptomycin (Sigma-Aldrich). CRISPR-cas9 edited parasites were supplemented with the appropriate selection antibiotics: BirA*::BDF5, KKT2::BirA*, BirA*::KKT3 or CLK2::BirA*: 4 μg ml⁻¹ puromycin (Invivogen), BDF5::mT and KKT3::mT: 4 μg ml⁻¹ puromycin, 10 μg ml⁻¹ blasticidin, loxP-flanked KKT24, KKT26: 4 μg ml⁻¹ puromycin, 10 μg ml⁻¹ blasticidin, 15 μg ml⁻¹ G418, 32 μg ml⁻¹ hygromycin B, mNG::KKT24, mNG::KKT26 KKT2::mCh: 4 μg ml⁻¹ puromycin, 10 μg ml⁻¹ blasticidin.

Cells were harvested in Western blot lysis buffer [100 mM tris-HCl (pH 8), 150 mM NaCl, 1% NP-40 substitute, and 1% protease inhibitor cocktail] for 30 min on ice. After brief sonication (5 cycles 30” ON/30” OFF, high output) with a standard Bioruptor (Diagenode), lysates were centrifuged at 13,000 rpm for 15 min at 4°C, and protein concentrations were determined using the Bradford assay (Bio-Rad). Proteins were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (Thermo Fisher Scientific) and transferred to nitrocellulose membranes (Thermo Fisher Scientific). Blots were incubated with primary antibodies (table S4) diluted in blocking solution (5% milk, 0.1% Tween 20 in PBS) overnight at 4°C followed by horseradish peroxidase–conjugated secondary antibodies (Cell Signaling). Blots were developed using SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. Densiometric analyses were performed with Fiji (v2.3.0). For the puromycin incorporation assay, cells were treated with puromycin (1 μg/ml; Invivogen) for 10 min, incubated with fresh medium for further 30 min, and then harvested.