Easy tag 35s protein labeling mix

Protein synthesis was assessed by labeling with ³⁵S-methinonine/³⁵S-cysteine over a 2 h period in the absence or the presence of 10x EC₅₀ of PA21A050 (10 nM), KAE609 (10 nM), cycloheximide (2000 nM), or artemisinin (100 nM). Briefly, parasite culture at 10% parasitemia was washed with methionine/cysteine free RPMI1640 supplemented with 0.5% Albumax. Labeling was done in 1 ml cultures at 5% hematocrit in methionine/cysteine free RPMI1640 containing 125 μCi/ml of Easy Tag ³⁵S Protein Labeling Mix (Perkin Elmer). After 1 h of incubation, 0.5 ml of regular cRPMI supplemented with 1 mg/ml of unlabeled methionine and cysteine plus the indicated amount of the compounds was added to the cultures, and incubation was continued for an additional 1 h. The cultures were divided into two 1.5 ml tubes, centrifuged, and washed twice with Albumax-free RPMI. Parasites were resuspended and treated with either 0.02% saponin or anthrolysin O as described above. Parasite pellets were resuspended in SDS-PAGE loading buffer, followed by SDS-PAGE and autoradiography.

SW1353 cells were incubated in 6-well plates for 30 min with 25 µCi/mL EasyTag [35S] protein labeling mix (Perkin Elmer, Rotterdam, The Netherlands) in DMEM without methionine or cystine, and supplemented with 10% FCS and 1% P/S. Cells were carefully washed four times with 0.9% NaCl and lysed in 150 µL RIPA buffer. Lysates were sonicated in a water bath and centrifuged for 15 min at 13,200 rpm in an Eppendorf tabletop centrifuge. A quantity of 50 µL of cleared lysates was measured for 5 min on a scintillation counter (Tri-Carb 2910 TR, Perkin Elmer, Rotterdam, The Netherlands), and data were extracted as counts per minute beta. Data were normalized to total DNA content per well using a Sybr Green DNA assay as described earlier [73 (link)]. Alternatively, SW1353 cells were incubated in a 96-well plate for 24 h with 50 µM of the methionine analogue homopropargylglycine (HPG) in DMEM without methionine and cystine, and supplemented with 63 mg/L cystine (Sigma-Aldrich), 10% FCS and 1% P/S. HPG incorporation was detected with a Click-IT reaction (HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit, Thermo Fisher Scientific, Breda, The Netherlands) with a TriStar2 (Berthold Technologies, Vilvoorde, Belgium). Data were normalized to total DNA content per well using the included HCS Nuclear mask blue stain.

Cells transfected with Tg variants were plated onto poly-D-lysine coated wells in 6-well dishes. Cells were incubated with methionine and cysteine depleted DMEM supplemented with glutamine, penicillin/streptomycin, and 10% FBS at 37 °C for 30 min. Cells were then metabolically labeled in DMEM depleted of methionine and cysteine and supplemented with EasyTag ³⁵S Protein Labeling Mix (Perkin Elmer, NEG772007MC), glutamine, penicillin/streptomycin, and 10% FBS at 37 °C for 30 min. Afterward, cells were washed twice with DMEM containing 10 X methionine and cysteine, followed by a burn off period of 30 min in normal DMEM. Cells were then chased for the respective time period with normal DMEM, lysed with 500uL of RIPA buffer with protease inhibitor cocktail and 10 mM DTT. Cell lysates were diluted with 500uL of RIPA buffer with protease inhibitor cocktail and subjected to immunoprecipitation with G1 anti-DYKDDDDK affinity resin overnight at 4° C. After three washes with RIPA buffer, protein samples were eluted with 3× Laemmli buffer with 100 mM DTT heating at 95 °C for 5 min. Eluted samples were then separated by SDS-PAGE, and gels were dried and exposed on a storage phosphor screen. Radioactive band intensity was then measured using a Typhoon Trio Imager (GE Healthcare) and quantified by densitometry in Image Lab (BioRad).