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. 2010 Jul 12;190(1):129–141. doi: 10.1083/jcb.200912087

Figure 2.

Figure 2.

SPSB2 interacts with full-length iNOS protein, and Tyr120 in the SPSB2 SPRY domain is critical for binding. (A) Overlay of the 1H-15N HSQC spectra of 0.1 mM 15N-labeled SPSB2 (12–224) in the absence (orange) and presence (green) of unlabeled iNOS peptide at SPSB2/iNOS peptide molar ratios of 1:1.5. Samples were in 95% H2O/5% 2H2O containing 10 mM Na phosphate, 50 mM Na chloride, 2 mM EDTA, 2 mM DTT, and 0.02% (wt/vol) Na azide at pH 7.0. Spectra were recorded at 500 MHz and 22°C. (B) Ribbon model of SPSB2 (12−224; PDB ID number 3EK9; Kuang et al., 2009) with residues whose 1H-15N cross-peaks experienced chemical shift perturbations upon iNOS peptide binding shown in pink. Helices, strands, and loops are colored green, gold, and purple, respectively. (C) BMDMs from C57BL/6 mice were incubated with 20 ng/ml IFN-γ and 1 µg/ml LPS overnight, lysed, and incubated with NHS-Sepharose beads coupled with recombinant SPSB2 or Y120A-SPSB2 protein or with uncoupled NHS-Sepharose beads (Bead) for 3 h at 4°C. Associated iNOS protein was detected by Western blotting (top). Equivalent amounts of SPSB2 and Y120A-SPSB2 were confirmed by reprobing with anti-SPSB2 antibodies (bottom). (D) 293T cells were transiently transfected with vector alone or cDNA encoding Flag-tagged SPSB1, SPSB2, SPSB3, or SPSB4. Cells were lysed and mixed with BMDM lysates from cells induced to express iNOS. Flag-tagged proteins were immunoprecipitated using anti-Flag antibodies, and coimmunoprecipitation of iNOS was detected by Western blotting (top). Membranes were stripped and reprobed with rat anti-Flag antibodies (middle). Comparative expression of Flag-tagged proteins in 293T lysates is shown in the bottom panel. (E) BMDMs from either wild-type (Spsb2+/+) or SPSB2-deficient mice (Spsb2−/−) were incubated with (+) or without (−) 20 ng/ml IFN-γ and 1 µg/ml LPS overnight and lysed, and endogenous SPSB2 proteins were immunoprecipitated using rabbit anti-SPSB2 antibody coupled to NHS-Sepharose. Associated iNOS protein was detected by Western blotting (top). Membranes were stripped and reprobed using biotinylated anti-SPSB2 antibody (middle). iNOS induction was confirmed by Western blotting of protein lysates (bottom). (F) 293T cells were transiently transfected with cDNA encoding Flag-tagged wild-type or mutant SPSB2 in which residues in the SPSB2 SPRY domain were mutated to Ala (and for Tyr120, also to Phe). Cells were lysed and mixed with BMDM lysates from cells induced to express iNOS. Flag-tagged proteins were immunoprecipitated using anti-Flag antibodies, and associated iNOS was detected by Western blotting. IP, immunoprecipitation.