Modulation of AAH, Humbug, and Junctin expression by growth factor stimulation. Subconfluent SH-Sy5y cell cultures were serum starved over night then stimulated with vehicle (CON), insulin (50 nM), IGF-1 (25 nM), or NGF (2.5 ng/ml) for 24 hours. (A) Upper panel: Representative Western blot demonstrating AAH protein expression (~86 kD) using the HBOH monoclonal antibody. Immunoreactivity was detected with horseradish peroxidase conjugated secondary antibody, ECL reagents, and digital imaging. Lower panel-blots were stripped and re-probed to detect β-actin as a loading control. The positions of molecular weight standards included in the analysis are indicated at the left. (B) AAH immunoreactivity was measured directly in 96-well micro-cultures using the microtiter immunocytochemical ELISA (MICE) assay. The MICE index corresponds to immunoreactivity corrected for cell density. (C) Directional motility was measured in response to insulin or IGF-I stimulation using the ATP Luminescence-Based Motility/Invasion (ALMI) assay. The total percentages of motile cells (motility index), both adherent and non-adherent, were calculated (see Methods). (D-F) AAH, Humbug, and Junctin mRNA levels were measured by real time quantitative RT-PCR with results normalized to 18S. See Methods section for detailed protocols. Graphs depict mean ± S.D. of results obtained from 6 or 8 replicate independent cultures. Data were analyzed using ANOVA with the Fisher Least Significant Difference post-hoc test (+P < 0.05; *P < 0.001 relative to control).