Fig. 1. Structure and expression of functionally active EGFP-RUNX fusion proteins. (A) A schematic of EGFP-RUNX expression constructs. EGFP proteins were fused to the N-termini of RUNX1, RUNX2 and RUNX2361. These constructs were generated using the restriction sites listed above each diagram as described in the Materials and Methods. EGFP-RUNX2361 lacks the C-terminal 152 amino acids of RUNX2 including the NMTS, but retains the RHD and NLS. Conserved functional domains of the fusion proteins are labeled as follows: EGFP: enhanced green fluorescent protein; QA: glutamine-alanine amino-acid stretch, specific to RUNX2; RHD: Runt homology domain; NLS: nuclear localization signal; NMTS: nuclear matrix targeting signal; VWRPY: conserved interacting sequence for TLE/Groucho, a repression protein. (B,C) Western blot analyses are shown of HeLa cell extracts after transfection with either EGFP-RUNX1, EGFP-RUNX2, EGFP-RUNX2361 or EGFP constructs. pcDNA3 empty vector, CMV-RUNX1 and CMV-RUNX2 expression vectors were used as controls. Proteins were detected using either a monoclonal EGFP antibody (B, top), a polyclonal RUNX1 antibody (C, right) or a polyclonal RUNX2 antibody (C, left). Cdk-2 was used as a control for equal protein loading (B and C, bottom panels). Positions of molecular weight markers are indicated on the right side of each blot. (D) CAT activity was assessed from HeLa cell extracts co-transfected with each reporter construct (OC promoter-CAT reporter and -83-OC-LUC) and each expression vector (EGFP, EGFP-RUNX2, EGFP-RUNX2361, RUNX2, EGFP-RUNX1 or RUNX1) as indicated. CAT values were normalized to the luciferase values and fold induction was calculated relative to the empty vectors. The results are means of 15 to 21 samples±s.e.m. P<0.0001.