Figure 2.

Expression of GP82 in transfected T. cruzi epimastigotes. (A) Three micrograms of proteins from total extracts of transfected epimastigotes were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes and incubated with mAb 9E10 (anti-c-myc), mAb 3F6 (anti-GP82) or anti-tubulin monoclonal antibodies. Samples were washed and incubated with peroxidase conjugated antibodies and the immunocomplexes developed by chemiluminescence. Void: transfected epimastigotes carrying the empty pTEX vector; 1^st: transfected parasites carrying the pTEX-1^st construct; 2^nd: epimastigotes transfected with pTEX-2^nd construct; M9/39L: epimastigotes transfected with pTEX-M9/39L construct. MT: wild-type metacyclic trypomastigotes from clone Dm28c or G strain. The vertical black line inside the panels denotes the boundary between lanes from the same developed membrane that were non-adjacent in the gel. Protein molecular weight standards (kDa) are indicated on the left. (B) Endoglycosidase H digestion of GPI-anchored enriched-protein extracts from transfected T. cruzi epimastigotes. GPI-enriched samples from transfected epimastigotes (5 × 10⁵ parasite/equivalents) and MTs (1 × 10⁵ parasite/equivalents) were treated (+) or mock treated (−) with 750 U of endoglycosidase H (Endo H) at 37 °C for 3 h. Samples were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes and incubated with anti-c-myc (upper panel) or anti-GP82 (lower panel) monoclonal antibodies. Immunocomplexes were developed as described in (A). Void: epimastigotes transfected with empty pTEX plasmid; 1^st: transfected parasites carrying the pTEX-1^st construct; 2^nd: epimastigotes transfected with pTEX-2^nd construct; M9/39L: transfected parasites carrying pTEX-M9/39L construct; MT: wild-type metacyclic trypomastigotes from clone Dm28c (1 × 10⁵ equivalent). Protein molecular weight standards (kDa) are indicated on the left. Full-length immunoblots are presented in Supplementary Fig. S5.