Fig. 4
p150 protein structure, as termed DPF, and expression patterns. a Predicted structure of protein p150 (DPF). Full-length DPF has an N-terminal signal peptide and C-terminal transmembrane domain. An antibody was to a specific peptide (Additional file 3: Figure S3C). The relative positions these features are positioned along the 1483 amino acid backbone. The N-FLAG DPF expression construct was created with a FLAG peptide sequence inserted in-frame, 3′ to the signal peptide. b Left panel—developmental expression of DPF mRNA at indicated times for WT cells, using RNA-blot hybridization. Right panel—relative DPF mRNA levels in growing WT cells, WT cells expressing full-length DPF (DPFOE), or WT cells expressing full-length FLAG-tagged DPF (N-FLAGOE), using RNA-blot hybridization. c Log-phase growing WT cells overexpressing DPF (DPFOE) were transferred into fresh growth media or fresh DB starvation buffer at similar cell densities and supernatant fractions taken and immunoblotted to α-DPF (see Fig. 4a). d Left panel—growing WT or WT cells expressing full-length FLAG-tagged DPF (N-FLAGOE) were transferred into fresh DB. Cell-free media were taken at times indicated and tested for relative density-dependent aggregation activity, using WT cells at 20 × 103 cells/cm2. Right panel—media collected at 7.5 h from both WT or N-FLAGOE cells were diluted into fresh DB, as indicated, and tested for relative density-dependent aggregation activity, using WT cells at 20 × 103 cells/cm2. Starting medium (1×) is 10-fold diluted, from a centricon concentrate of conditioned supernatant. e Log-phase growing WT cells were plated under DB buffer at 25 × 103 cells/cm2 for 8 h, using either fresh, naïve DB media, or cell-free, > 30-kDa conditioned media from WT or DPFOE cells starved in DB for 5 h (see Fig. 4d,e). Relative density-dependent activity is indicated as % aggregation. Scale = 400 μm