Figure 1.

Identification of a Gα12-interacting region in AKAP-Lbc. (A) Sequence alignment of AKAP-Lbc with other Gα12 signaling targets. Results of Expasy SIM alignment using amino acid sequences of human proteins AKAP-Lbc (GenBank: NP_006729), axin-1 (NP_003493), and RGS1 (AAH15510) are shown. Regions of AKAP-Lbc excluding the tandem DH/PH domains were examined for homology to short sequences within RGS-1 and axin. Black vertical dashes indicate identical residues, open dashes indicate residues with similar properties. (B) Binding of Gα12 to an AKAP-Lbc region similar to axin and RGS1. HEK293 cells were transfected with a plasmid encoding myc-tagged, constitutively active Gα12 (GTPase-deficient Q229L mutant), or with pcDNA3.1 vector, and detergent-soluble extracts prepared as described in Methods. For each transfected sample, 5% of diluted extract was set aside as starting material (Load), and co-precipitation assays were performed using a Sepharose-bound GST-fusion of a 257-residue region of AKAP-Lbc, or GST alone. After SDS-PAGE and electroblot transfer, nitrocellulose membranes were probed with anti-Gα12 antibody (Santa Cruz Biotechnology; sc-409). Immunoblot images shown are representative of >5 experiments, with bands visible at the expected size (~45 kDa) for myc-tagged Gα12 and not detected in vector-transfected samples.