1. LACK/LACKDDE L. major show robust nascent global protein synthesis at 27 and 35°C.
LACK/LACKDDE L. major show robust nascent global protein synthesis at 27 and 35°C. Logarithmically growing parasites were established at 27 and 35°C, as described in Fig. 4 and 5, respectively. (A) After 3 h of incubation with azidohomoalanine (AHA) in methionine-free RPMI 1640 medium–10% dialyzed FBS, parasites were lysed, and nascent protein synthesis was detected via click-chemistry coupling to TAMRA, followed by SDS-PAGE electrophoresis and imaging using a Typhoon TRIO+ variable mode Imager (GE) with a 532-nm excitation and 580-nm 30-nm band pass filter. As a negative control, we also included lysate from parasites that were treated with cycloheximide (CHX) immediately prior to addition of AHA and incubation for 3 h. The image shown is representative of two independent experiments, each performed with a different clone of each line, as indicated. (B) After imaging for nascent protein synthesis, the total protein present in each lane was assessed by silver staining. The data shown are from one experiment, representative of two independent experiments, each with different clones, performed in duplicate. (C) Average total imaged band intensities in each lane were quantified from the two independent experiments performed in duplicate, using Typhoon/Molecular Dynamics analysis software (GE). Protein size markers (kDa) are indicated.
Daviel Cardenas et al. mSphere 2019; doi: /mSphere