CCN3 protein expression is down-regulated as a result of BCR-ABL kinase activity. (A) FDCP-Mix control and ts-BCR-ABL cells were grown at the restrictive temperature (39°C) and permissive temperature for BCR-ABL activity (32°C) for 24 hours. Protein lysates were extracted and probed for CCN3 and actin expression; levels of expression corrected for protein loading are shown as integrated optical densitometry units (IODs). ^*P = .026; n = 3. (B) Confocal microscopy was also used to identify CCN3 protein (green) in FDCP-Mix control cells at 39°C (i) and 32°C (ii) and ts-BCR-ABL cells at 39°C (iii) and 32°C (iv); propidium iodide nuclear staining (red) is also shown. Images were collected using a Bio-Rad Microradiance confocal laser scanning microscope with an oil immersion lens (magnification, × 160). (C) Medium in which FDCP-Mix control and ts BCR-ABL cells were grown for 24 hours was collected and probed for secreted CCN3; levels of secreted CCN3 are shown as IODs for comparison. ^*P = .014; n = 3. (D) Lysates from control cells and BCR-ABL kinase-active cells grown at the permissive temperature (32°C) were immunoprecipitated using CCN3 antibody and probed for (i) phosphotyrosine, (ii) CCN3, and (iii) actin expression. Levels of CCN3 and phosphorylated CCN3 detected by Western blot analysis in FDCP-Mix control and BCR-ABL kinase-active cells were compared using densitometry as shown.