Figure 1
From: Characterization of the 26S proteasome network in Plasmodium falciparum
Identification of ubiquitin receptor domains in P. falciparum 26S proteasome components.
(A,B) Assessment of the capability of PfUIM and PfPru domains in binding K48-linked (A) or K63-linked (B) polyubiquitin in a Ni-NTA pull-down assay. The His-tagged PfUIM domains, PfPru domain and PfTrx1 (as a control) of equal molar concentrations (4.5 μM) were individually incubated with 5 μg K48-linked or K63-linked polyubiquitin of chain length ranging from one to seven (Ub1–7) in a Ni-NTA pull-down assay. Ubiquitin was probed by immunoblotting using an anti-ubiquitin antibody. (C) Association of His-tagged receptor domains with endogenous ubiquitinated proteins was examined by a Ni-NTA pull-down assay using P. falciparum extracts. The pulled-down ubiquitin-conjugates were detected by immunoblotting using an anti-ubiquitin antibody. The blotting membrane was stained with Ponceau S to show the amount of bait proteins. (D) Binding PfUIM and PfPru domains to UBL domains of PfRad23 and PfDsk2. The GST-fused UBL domains of PfRad23 and PfDsk2 were pulled down by the respective PfUIM and PfPru domains but not by PfTrx1 (as a bait control), as detected by immunoblotting using an anti-GST antibody. The blotting membrane was stained by Ponceau S to show the amount of bait proteins. GST itself is hardly pulled down by PfUIM and PfPru domains. IB, immunoblotting. Ub, ubiquitin.
