The interaction between Tks proteins and NoxA1 is direct. (A) GST-fused organizer subunit proteins Tks4, Tks5 and NoxO1 were bound to glutathione-Sepharose beads and challenged with appropriate amounts of GST-cleaved recombinant NoxA1 protein. GST-pulldown was performed as described in Materials and methods. Bound proteins were resolved by SDS-PAGE and probed with NoxA1 antibody. One representative experiment from three separate experiments is shown. (B) The reverse pulldown approach confirms that the interaction between Tks proteins and NoxA1 is direct. GST-fused organizer subunit proteins Tks4, Tks5 and NoxO1 were incubated with appropriate amounts of His-tagged NoxA1 protein. The pulldown was performed using Talon magnetic beads (see Material and methods). Bound proteins were resolved by SDS-PAGE and probed with GST antibody. One representative experiment from three separate experiments is shown.

Schematic representation and expression of Myc- and GST-tagged NoxA1 deletion mutants. (A, B) Schematic representation of Myc-tagged (A) and GST-tagged (B) NoxA1 deletion mutants which we have used to identify the NoxA1 region responsible for the interaction with Tks proteins. Rectangles indicate N-terminal Myc (A) or GST (B) tag. Triangles indicate Proline-Rich Regions (PRRs). Octagons indicate tetratricopeptide (TPR) domains. Crosses indicate “Phox and Bem 1” (PB1) domain, while ovals indicate Src Homolgy-3 (SH3) domains. (C) Myc-tagged NoxA1 deletion mutants produce a protein of the expected molecular size when transfected into HEK293 cells. HEK293 cells were transfected as indicated with Myc-tagged NoxA1 deletion mutants and after 24 hrs their expression was determined by Western blot as described in Materials and methods using Myc antibody. (D) GST-tagged NoxA1 deletion mutants exhibit the expected molecular size when expressed and purified in E.Coli as described in Materials and methods. Purified proteins were then resolved by SDS-PAGE, transferred onto a nitrocellulose membrane and the molecular size of the purified truncated and wild-type proteins (indicated by black arrows) was checked by staining the membrane with S-Ponceau.

The N-terminal region of NoxA1 is responsible for the interaction with Tks proteins. (A, B) Co-immunoprecipitation analysis of Myc-tagged NoxA1 deletion mutants indicates that the first 155 amino acid residues of NoxA1 are involved in the interaction with Tks5. One representative experiment from three separate experiments is shown for each. (A) HEK293 cells were transfected as indicated with Myc-tagged NoxA1 deletion mutants and with Flag-tagged Tks5. After 24 hrs, cells were lysed and immunoprecipitation (IP) was carried out (see Material and methods) using Myc antibody. The interaction between Myc-tagged NoxA1 deletion mutants and Tks5 was tested by immunoblot (IB) using Tks5 antibody. Lys indicates lysate before IP was performed. (B) The expression level of all transfected Myc-tagged NoxA1 deletion mutants was analyzed by Western blot using Myc antibody. (C, D) GST-pulldown analysis confirms that the N-terminal region of NoxA1 is involved in the binding with Tks4. One representative experiment from three separate experiments is shown for each. (C) Cell lysates from human DLD1 colon cancer cells were used as the source of Tks4 protein and incubated as indicated with equal amounts of GST alone or GST-fusion NoxA1 truncated or full length proteins, which were pre-bound to glutathione-Sepharose beads. GST-pulldown was performed as described in Material and methods and the interaction between GST-fusion NoxA1 proteins and endogenous Tks4 was tested using the Tks4 antibody. (D) Ponceau S stain of nitrocellulose membrane from GST-pulldown experiment performed in C shows that the GST-fusion proteins (indicated by black arrows) were present at similar levels in the GST-pulldown analysis.

The integrity of PRR (34–37) of NoxA1 is necessary for the interaction with Tks5 and for Tks5-mediated ROS generation by Nox1. (A) Schematic representation of domain composition (upper panel) and amino acid sequence alignment (lower panel) of activator subunits NoxA1 and p67^phox. The highlighted light gray sequences indicate the conserved PRR between NoxA1 and p67^phox, while the highlighted dark gray sequence indicates the N-terminal non-conserved PRR lost in p67^phox. (B) Co-immunoprecipitation analysis reveals that Tks proteins cannot bind p67^phox. HEK293 cells were transfected as indicated with p67^phox and either Myc-tagged Tks4, Tks5 or p47^phox. After 24 hrs, cells were lysed and immunoprecipitation (IP) was carried out using Myc antibody. The interaction between Myc-tagged organizer subunits and p67^phox was tested by immunoblot (IB) using p67 antibody (upper panel), while comparable expression levels of transfected Myc-tagged organizers in cell lysates and immunoprecipitation efficiency was assessed by re-blotting the membrane with Myc antibody (lower panel). One representative experiment from three separate experiments is shown. (C) Co-immunoprecipitation analysis indicates that the PRR (34–37) of NoxA1 is involved in its interaction with Tks5. HEK293 cells were transfected as indicated with NoxA1 AXXA mutants, NoxA1 wild type and with Flag-tagged Tks5. After 24 hrs, cells were lysed and immunoprecipitation was carried out using Flag antibody. Similar expression levels of Flag-tagged Tks5 and NoxA1 AXXA mutants in the lysates was tested using Tks5 and NoxA1 antibody respectively (upper panels). The interaction between NoxA1 AXXA mutants and Tks5 was tested using NoxA1 antibody (lower panel, top), while comparable immunoprecipitation efficiency was controlled by re-blotting the membranes with Tks5 antibody (lower panel, bottom). One representative experiment from three separate experiments is shown. (D) The NoxA1 AXXA (34–37) blocks Tks5-mediated, Nox1-dependent ROS generation compared with wild-type NoxA1. HEK293 cells were co-transfected as indicated with empty vector or with Nox1, Rac1-Q61L and Tks5 expression plasmids along with either empty vector, wild type NoxA1 or NoxA1 AXXA (34–37). After 24 hrs, ROS production was determined using the luminol-based chemiluminescence assay. One representative experiment from three separate experiments is shown, and data are given as mean of triplicates +/− S.D. * p<0.01 compared to the condition with wild type NoxA1.