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. Author manuscript; available in PMC: 2012 Mar 29.
Published in final edited form as: Biochemistry. 2011 Mar 4;50(12):2013–2025. doi: 10.1021/bi1020088

Figure 4. Oligomerization is mediated by the Nox5 dehydrogenase domain.

Figure 4

(A) Schematic diagram of full-length of CiVSP (CiVSP), full-length Nox5 (Nox5) and a chimera comprised of the transmembrane (TM) portion of CiVSP fused to the Nox5 DH domain (chimera, CiVSP-N/Nox5-DH-C). Abbreviations are: N-tail, N-terminal cytoplasmic tail region of CiVSP; 4TM, the four-helix transmembrane region of CiVSP; Ptase, the phosphatase domain of CiVSP. Other abbreviations are as in the legend to Figure 3A. (B) Equal amounts (5 μg) of the indicated vector were cotransfected into HEK293 cells. Lysates were immunoprecipitated using an anti-myc monoclonal antibody, resolved by SDS-PAGE and immunoblotted with an anti-GFP antibody (top panel) and an anti-myc antibody (middle panel). * is the IgG heavy chain. Italic letters (a–f) refer to immunostaining protein bands [a, GFP-CiVSP; b, GFP-chimera (CiVSP-N/Nox5-DH-C); c, GFP-Nox5; d and e, chimera-myc (CiVSP-N/Nox5-DH-C); f, Nox5-myc]. A protein band appeared around 77 kDa in lane 4 (**) is the degradative product of GFP-Nox5 protein. To evaluate the expression of GFP-tagged proteins in the cell lysate, GFP-fused proteins were immunoprecipitated using anti-GFP monoclonal antibody, resolved by SDS-PAGE and immunoblotted using anti-GFP antibody (bottom panel). Similar results were obtained in four independent experiments. (C) ROS production in HEK293 cells co-transfected with empty vector (mock), full-length, N-terminally hexahistidine-tagged Nox5 (Nox5-WT), and full-length, N-terminally GFP-tagged CiVSP (GFP-CiVSP) or the chimera consisting of the N-terminally GFP-tagged CiVSP TM domain linked to the Nox5 DH domain [GFP-chimera (CiVSP-N/Nox5-DH-C)] was measured using luminol luminescence. Equal expression of full-length hexahistidine-tagged Nox5 (Nox5-WT) in cell lysates (CL) was evaluated using immunoblotting with an anti-hexahistidine-tag antibody (α-His, bottom), and protein loading was assessed with immunoblots using antibody to β-actin. Data are the mean ± SD (n = 4); *, P < 0.05, **, P < 0.01, compared with Nox5 WT + GFP. Similar results were obtained in two independent experiments.