Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein
Figure 5
Expression of exogenous EhVps4 in transfected trophozoites. (a) Schematic representation of pEhVps4-E211Q construct. Mutant Ehvps4 gene was cloned into the KpnI and BamHI sites of pNEO vector, upstream the sequence coding for FLAG epitope. B, BamHI site; K, KpnI site. (b) 2% agarose gel with RT-PCR assays products. An internal fragment of the Ehvps4 gene (upper panel) or the Eh25S rRNA gene (bottom panel) was RT-PCR amplified using 1 g of total RNA from trophozoites of clone A transfected with pNEO (lane 1), pEhVps4 (lane 2) or pEhVps4-E211Q (lane 3) plasmid. (c) Western blot assays. Anti-FLAG (upper panel), anti-rEhVps4 (middle panel) and anti-actin (lower panel) antibodies were used to analyze protein lysates (30 g) from pNEO (lane 1), pEhVps4 (lane 2) and pEhVps4-E211Q (lane 3) transfected cells. (d) Densitometric analysis of bands corresponding to actin and EhVps4 in (c). Pixels corresponding to actin control were taken as 100% in each lane and used to normalize EhVps4 data.
