Northern blot analysis of OPN gene expression. (A) Northern blot of peripheral blood monocytes (lane 1) and 3-day cultures of HS-27a alone (lane 2) and adherent cells from cocultures of HS-27a and monocytes with different monocyte-HS-27a ratios (lanes 3-5). The monocyte-HS-27a ratios at day 0 in lanes 3, 4, and 5 are 1:20, 1:6, and 1:2, respectively. Total RNA (5 μg per lane) was separated on an RNA agarose gel and blotted onto a nylon membrane. The membrane was probed with a ³²P-labeled OPN probe. The bound probe was stripped off, and the membrane was reprobed with a ³²P-labeled actin probe. (B) Quantitation of the Northern blot by phosphor image analysis showed a strong correlation between the ratio of monocytes to HS-27a and OPN gene expression (R² = 0.99). The data indicate a strong signal for OPN in RNA isolated from cocultures but not HS-27a or CD14⁺ cells cultured alone.