Figure 2
From: Characterization of the 26S proteasome network in Plasmodium falciparum
Analyses of 26S proteasome complexes of P. falciparum.
(A) Native gel electrophoresis of P. falciparum extracts followed by an in-gel proteasome activity assay with chymotrypsin-like activity substrate Suc-LLVY-AMC. CP, 20S proteasomes; RP1CP, singly 19S-capped 20S proteasomes; RP2CP, doubly 19S-capped 20S proteasomes. (B) Workflow of the affinity purification of P. falciparum 26S proteasomes using a GST-UBL-based strategy. See Experimental procedures for more details. (C) Proteins in affinity-purified proteasome samples were resolved via 12% SDS-PAGE followed by silver staining analysis. A control sample from a GST-based purification was analyzed in parallel. The presence of the 20S subunits in the purified proteasome sample was confirmed by immunoblotting with an antibody (MCP231) recognizing plasmodial 20S α-subunits. (D) The specific chymotrypsin-like, trypsin-like and PGPH activities of the affinity-purified P. falciparum proteasomes in the absence or presence of MG132 and lactacystin (10 μM) were individually determined by using the fluorogenic substrates Suc-LLVY-AMC, Bz-VGR-AMC and Z-LLE-AMC, respectively. Each value is a mean ± S.D. from at least three independent purifications. No proteasomal activity was observed in the samples obtained from the GST-based purification.
