Article Figures & Data
Figures
- FIG. 1.
Noxo1-dependent and Rac1-independent subcellular localization of Noxa1 in HEK293 cells. A. Noxo1-GFP is detected predominantly along the plasma membrane. Coexpressed Noxa1 colocalizes with Noxo1 along the plasma membrane. B. The association of Noxa1 with the plasma membrane is disrupted by an SH3 domain mutation (W436R) that was shown to disrupt its interaction with the C-terminal, proline-rich region of Noxo1. C. Noxa1 expressed alone is localized primarily in the cytosol, although some Noxa1 accumulates at ruffling membranes (arrow). D. Localization of Noxa1 at ruffling membranes is not affected by a mutation [Noxa1(R103E)] that disrupts its interaction with Rac1. E. The Noxa1 antibody detects transfected Noxa1 as a doublet band around 50 kDa. Wild-type Noxa1 is detected in both cytosol and membrane fractions, consistent with fluorescence imaging. Membrane-associated Noxa1 increases by coexpression of Noxo1-GFP but not Noxo1(W436R)-GFP. Plasma membrane localizing protein, Noxo1-GFP, is detected predominantly in the membrane fraction. F. The fractionation of Noxa1 is not affected by a Noxa1 mutation at R103E or coexpression of Rac1(Q61L). Right, cytosolic (unfused) GFP is detected predominantly in the cytosol fraction. G. Fractionation of Noxa1 is not affected by two Rac1-suppressing siRNAs (Stealth and Silencer). Immunoblotting of total lysates confirms effective suppression of endogenous Rac1 by these siRNAs. no siRNA, transfection without siRNA. Bar, 10 μm. Similar protein loading is confirmed by β-tubulin blotting. Similar results were obtained in four (A, B, C, D, and F) or three (E and G) independent experiments.
- FIG. 2.
Nox1 activity supported by membrane-targeted Noxa1 is Rac dependent but Noxo1 independent. A. Optimal reconstitution of Nox1 activity in HEK293 cells requires cotransfection with partners Noxo1 and Noxa1. Cotransfection of Nox1 with Noxo1, Noxa1, or constitutively active Rac1(Q61L) does not support significant activity. Membrane-targeted Noxa1(pp) can partially compensate for the absence of Noxo1; coexpression of Rac1(Q61L) dramatically enhances Nox1 activity supported by Noxa1(pp). Data are from at least three independent transfection experiments. B. Schematic representation of Noxa1(pp) structural features. C. Noxa1(pp) accumulates along the plasma membrane. Bar, 10 μm. Right, cell fractionation confirms Noxa1(pp) localization in the membranes; similar results were obtained in two independent experiments. D. Nox1 activity supported by Noxa1 or Noxa1(pp) is enhanced by Rac1(Q61L) and effectively inhibited by the Rac binding site mutation (R103E). Data are from at least three independent transfection experiments. E. Transfected Noxa1 proteins are detected by Western blotting at comparable levels. Similar protein loading is confirmed by β-tubulin blotting.
- FIG. 3.
Rac1 involvement in the fully activated Nox1 system. A. Neither Rac1(G30S) nor Rac1(Q61L) affects Nox1 activity in the HEK293 cell model. Data are from at least three independent transfection experiments. B. Rac1(G30S) inhibits both basal and PMA-stimulated Nox1 activity in transfected CHO-K1 cells, while Rac1(Q61L) enhances basal activity. Data represent means ± SD of at least three independent transfection experiments. (*, P < 0.05, compared with no Rac1 transfection [pcDNA3.1]). C. Western blotting of CHO-K1 cell lysates detects both the endogenous and Myc-tagged Rac1 proteins. Comparable protein loading is confirmed by β-tubulin blotting. D. Rac1(G30S) inhibits Nox2 activity in transfected HEK293 and CHO-K1 cells, while Rac1(T17N) inhibits Nox2 only in CHO-K1 cells. Data represent means ± SD of at least three independent transfection experiments. (*, P < 0.05, compared with no Rac1 transfection [pcDNA3.1]). Western blotting (top panels) detects endogenous and the mutant Myc-tagged Rac1 proteins. E. Immunofluorescence detection of endogenous Rac1 in HEK293 cells reveals significant Rac1 accumulation along the plasma membrane, while Rac1 is localized primarily to the cytosol in CHO-K1 cells. Bar, 10 μm. F. Rac1 activation assay detects activated endogenous Rac1 (GTP bound) in unstimulated HEK293 cells but not in CHO-K1 cells. Similar results were obtained in two independent experiments.
- FIG. 4.
Suppression of Nox1 activity by Rac1-specific siRNA. A. Transfection with pSUPER-Rac1-147, pSUPER-Rac1-681, or pSUPER-Rac1-1664 but not pSUPER Rac1-320 results in significant knockdown of endogenous Rac1 protein levels compared with control plasmid lacking RNAi sequence [pSUPER (vector)] in HEK293 cells. Similar protein loading is confirmed by β-tubulin blotting. B. Unstimulated Nox1 activity supported by Noxa1(pp) is significantly inhibited by pSUPER-Rac1-147, pSUPER-Rac1-681, or pSUPER-Rac1-1664 in HEK293 cells. Data represent means ± SD of at least three independent transfection experiments [*, P < 0.05, compared with pSUPER (vector)]. C. Unstimulated Nox1 activity supported by Noxo1 and Noxa1 is also inhibited by Rac1 Stealth siRNA or Rac1 Silencer siRNA in HEK293 cells, and reintroduction of wild-type human Rac1 (0.5 μg) restores Nox1 activity suppressed by Rac1 Silencer siRNA (both unstimulated and PMA stimulated). Right, Western blotting confirms significant suppression of endogenous Rac1 protein by Rac1 Silencer siRNA and reintroduction of Myc-tagged wild-type human Rac1 (lysates are from the same number of cells). Data represent means ± SD of at least three independent transfection experiments (*, P < 0.05, compared with no siRNA and negative-control siRNA). D. Cotransfection of Noxo1 and Noxa1 enhances basal and PMA-stimulated ROS production by HT-29 cells. These activities are further enhanced by Rac1(Q61L). ROS production supported by Noxo1 and Noxa1 is significantly inhibited by Rac1 Validated Stealth siRNA and is restored by reintroduction of wild-type mouse Rac1. Data represent means ± SD of at least three independent transfection experiments (*, P < 0.05, compared with negative-control siRNA). E. Western blotting of HT-29 cell lysates confirms expression of Rac1(Q61L), suppression of endogenous Rac1 protein by Rac1 Stealth siRNA, and reintroduction of GFP-tagged wild-type mouse Rac1 (lysates are from the same number of cells, and similar protein loading is further confirmed by β-tubulin blotting). F. Transfected vector-based RNAi (pSUPER-Rac1-681) and synthetic Rac1 siRNA (Stealth siRNA) are also effective inhibitors of the Nox2 system in HEK293 cells. Data represent means ± SD of at least three independent transfection experiments [*, P < 0.05, compared with no siRNA and negative-control siRNA or pSUPER (vector)].
- FIG. 5.
Disruption of the fully activated Nox1 system by mutations affecting interactions with Rac1, Noxo1, Noxa1, and p22phox. A. The fully activated Nox1 system, supported by Noxo1 and Noxa1, is only partially affected by Noxa1(R103E), Noxa1(W436R), or Noxo1(ΔPRR) mutations, which disrupt interactions with Rac1, Noxo1. or Noxa1, respectively. Combined disruption of Noxa1-Rac1 and Noxo1-Noxa1 interactions completely inhibits the Nox1 system, as does a disruption of the Noxo1-p22phox interaction. Data are from at least three independent transfection experiments. B. Schematic representation of sites that effectively disrupt Nox1 activity, involving mutations in Noxa1 (left) or Noxo1 (right). The panel is adapted from reference 22. C. Western blotting of HEK293 cell lysates confirms comparable expression of Noxo1-V5 mutants. D. Western blotting confirms comparable expression of Noxa1 mutants. Lower (control) panels confirm comparable protein loading by β-tubulin blotting.
- FIG. 6.
Nox1- and Nox3-dependent targeting of p22phox to the plasma membrane. A and B. Immunofluorescence imaging of endogenous (A) and transfected (B) p22phox in HEK293 cells, showing reticular cytosolic and nuclear membrane (arrows) staining patterns. C. Transfection of Nox1 results in a redistribution of endogenous p22phox to the plasma membrane. D. A similar redistribution of p22phox occurs in Nox3-transfected cells. *, untransfected cells showing primarily cytosolic staining patterns and overall weaker staining.
- FIG. 7.
Nox3 activity supported by Noxa1 or p67phox is Rac1 dependent. A and B. Reconstitution of Nox3 activity with Nox proteins (Noxo1 or Noxa1) in HEK293 and CHO-K1 cells. Noxo1-supported activity is abolished by the SH3 domain (p22phox binding site) mutation. Noxa1-supported activity is enhanced by Rac1(Q61L) and abolished by the Rac1 binding site mutation (R103E). Nox3 activity supported by membrane-targeted Noxa1 is inhibited by the R103E mutation. Data are from at least three independent transfection experiments. C and D. Reconstitution of Nox3 activity with phox proteins in HEK293 and CHO-K1 cells. The p47phox-supported Nox3 activity is inhibited by the p22phox binding site mutation (W193R), while Nox3 activity supported by p67phox (wild-type and membrane-targeted forms) is inhibited by the Rac1 binding site mutation. Data are from at least three independent transfection experiments. E. Western blotting of HEK293 cell lysates detects comparable levels of p47phox (left) and p67phox (right) mutant proteins. Comparable protein loading is confirmed by β-tubulin blotting.
- FIG. 8.
Suppression of Nox3 activity by Rac1-specific siRNAs in HEK293 cells. A. Nox3 basal activity, as well as Nox3 activity supported by Noxa1, p67phox, or p67phox and p47phox, is significantly inhibited by pSUPER-Rac1-147, pSUPER-Rac1-681, or pSUPER-Rac1-1664 (vector-based RNAi). Data represent means ± SD of at least three independent transfection experiments [*, P < 0.05, compared with pSUPER (vector)]. B. Nox3 activity supported by p67phox and p47phox is significantly inhibited by Rac1 Stealth siRNA or Rac1 Silencer siRNA and restored by reintroduction of wild-type human Rac1 (0.5 μg). Right panel, Western blotting confirms significant knockdown of endogenous Rac1 protein by Rac1 Silencer siRNA and production of Myc-tagged wild-type human Rac1 (lysates are from the same number of cells). Data represent means ± SD of at least three independent transfection experiments (*, P < 0.05, compared with no siRNA and negative-control siRNA).
- FIG. 9.
p67phox localizes to ruffling membranes in HEK293 cells. A. p67phox is detected in cytosol and ruffling membranes; Rac1 colocalizes with p67phox on membrane ruffles. B. p67phox(R102E) also colocalizes with Rac1 within ruffling membranes. C. Wild-type and p67phox(R103E) are distributed similarly between cytosol and membrane fractions. Right, fractionation of p67phox is not affected by two Rac1-specific siRNAs. Immunoblotting of total lysates shows effective knockdown of endogenous Rac1 by the Rac1-specific siRNAs. no siRNA, transfection without siRNA. D. Schematic representation of the p67phox(pp) structure. E. Plasma membrane targeting of p67phox(pp). Right, fractionation study confirms p67phox(pp) targeting to the membrane fraction. Bar, 10 μm. Comparable protein loading is confirmed by β-tubulin blotting. Similar results were obtained in four (A, B, C [left], and E [left]), three (C [right]), or two (E [right]) separate experiments.
