Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein
Figure 3
Expression and immunodetection of EhVps4. (a) Expression and purification of recombinant EhVps4-GST polypeptide. Proteins were separated through 10% SDS-PAGE and gels were stained with Coomassie blue. Lane 1, molecular weight markers; lane 2, noninduced bacteria extract; lane 3, IPTG-induced bacteria extract; lane 4, affinity purified rEhVps4-GST from IPTG-induced bacteria extract. (b) Immunodetection of rEhVps4-GST. Western blot assays were performed using noninduced bacteria extract (lane 1) and IPTG-induced bacteria extract (lane 2), with anti-GST antibodies. (c) PreScission Protease digestion of rEhVps4-GST revealed by Coomassie blue stained gels. Lane 1, molecular weight markers; lane 2, purified rEhVps4-GST protein; lane 3, rEhVps4-GST digested with PreScission Protease; lane 4, purified rEhVps4 protein. (d) Immunodetection of purified rEhVps4 protein by Western blot assays using specific anti-rEhVps4 antibodies (lane 1). (e) Immunodetection of EhVps4 in total extracts of trophozoites using specific anti-rEhVps4 antibodies (lane 1) and preimmune serum (lane 2). Proteins are indicated by arrowheads. (f) and (g) Cellular localization of EhVps4 in trophozoites. Trophozoites of clone A were incubated with rabbit anti-rEhVps4 (f) or preimmune (g) serum, treated with FITC-labeled secondary antibodies, counterstained with DAPI and analyzed through confocal laser microscopy. Left, cells observed in phase contrast; right, merge (trophozoites observed in the green (FITC) and blue (DAPI) channels). Arrowheads, EhVps4 signal in small cytoplasmic dots.
