Figure 1. CAPERα and TBX3 directly interact via the TBX3 repressor domain.
(A) Representative spectrum for CAPERα identified in anti-TBX3 co-IP of HEK293 cell lysates. Mass spec analysis identified six specific CAPERα peptides, providing 8.5% sequence coverage of the protein. This spectrum shows fragmentation of one of these peptides, C*PSIAAAIAAVNALHGR, with diagnostic b- and y-series ions shown in red and blue, respectively. * indicates carbamidomethylation. (B) Anti-CAPERα immunoblot (IB) analysis of anti-CAPERα immunoprecipitated (IP'd, lane 2) e10.5 mouse embryo lysates. Black arrowheads indicate IgG heavy chain and red indicate protein of interest (CAPERα or TBX3). (C) Anti-Tbx3 IB of anti-Tbx3 (lane 4) and anti-Caperα (lane 5) IP'd mouse embryo lysates. Rabbit (r)-IgG (lanes1, 6) and mouse (m)-IgG (lane 7) are negative controls. (D) In vitro MBP pull down assay: MBP and MBP-Tbx3 bound amylose affinity columns were incubated with GST or GST-CAPERα. Bound proteins were eluted, subjected to SDS-PAGE followed by IB with anti-CAPERα antibody. (E–G) Colocalization of Tbx3 and Caperα in vivo shown by immunohistochemical analysis of sectioned e10.5 mouse embryo: embryonic dorsal root ganglion (DRG, E), proximal (F), and distal (G) limb bud with anti-Tbx3 (red) and anti-Caperα (green) antibodies and DAPI (blue). White arrowheads in G label representative ectodermal and mesenchymal cells with cytoplasmic Tbx3 and nuclear Caperα. (H) Schematic representation of mouse Tbx3 overexpression constructs.Tbx3 DNA binding domain (DBD) point, ΔRD and exon7 missense proteins are untagged and the C-terminal deletion mutants are Myc-tagged. (I) Anti-TBX3 IB of HEK293 cell lysates transfected with control or anti-TBX3 shRNA. (J) Anti-CAPERα IB of anti-CAPERα IP'd samples from HEK293 cells transfected with anti-TBX3 shRNA and expressing mouse Tbx3 proteins listed at top. Production and IP of endogenous CAPERα is not affected by production of mutant Tbx3 proteins. (J′) Anti-Tbx3 IB of anti-CAPERα IP'd samples from HEK293 cells transfected with anti-TBX3 shRNA and expressing Tbx3 proteins as in J. The DBD point mutant proteins (lanes 2, 3) interact with CAPERα as efficiently as wild type Tbx3 (lanes 1, 4). (K) Anti-Myc IB of anti-Myc IP'd samples from HEK293 cell lysates expressing Myc-tagged mouse Tbx3 C-terminal deletion mutants. The mutant proteins are expressed and efficiently IP'd. These cells were not treated with anti-TBX3 shRNA because the expression constructs produce a Myc- tagged mutants that can be IP'd independently of endogenous TBX3. (K′) anti-CAPERα IB of anti-Myc IP'd samples from HEK293 cell lysates expressing Myc-tagged mouse Tbx3 C-terminal deletion mutants. These cells were not treated with anti-TBX3 shRNA because the expression constructs produce a Myc- tagged mutants that can be IP'd independently of endogenous TBX3. (L) Anti-Tbx3 IB of anti-Tbx3 IP'd samples from HEK293 cells transfected with anti-TBX3 shRNA and expressing wt or repressor domain deletion mutant (ΔRD) mouseTbx3. The shRNA does not prevent production of the overexpression proteins. (L′) Anti-CAPERα IB of HEK293 cells transfected with anti-TBX3 shRNA and expressing mouse wt or ΔRD Tbx3 proteins and IP'd with anti-Tbx3 or IgG. Loss of the repressor domain prevents interaction with CAPERα. Black arrowheads indicate IgG heavy chain and red indicate protein of interest (CAPERα or TBX3). TBX3, CAPERα = human; Tbx3, Caperα = mouse.
Figure 1—figure supplement 1. Missense mutation of the C-terminus of Tbx3 disrupts interaction with CAPERα.
