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. 1998 Feb 17;95(4):1788–1793. doi: 10.1073/pnas.95.4.1788

Figure 1.

Figure 1

Northern analysis of poly(A) mRNA for COX-1 and COX-2 in HIT-T15 (H; lanes 1–3) cells grown in RPMI 1640 medium containing 10% FBS. Twenty-eight hours before harvesting cells for RNA extraction, FBS was removed from the medium. Cells were incubated in media with 0%, 0.2%, or 10% FBS for 4 hr immediately before harvesting. Total RNA was extracted and poly(A) mRNA was isolated. Six micrograms of poly(A) mRNA from HIT cells were loaded per lane, electrophoresed in a 1.5% agarose-formaldehyde gel, and transferred to a nylon membrane by electroblotting. Poly(A) mRNA was used for these experiments because the size of COX-2 mRNA is close to the size of 28 S ribosomal RNA. Twenty micrograms of total RNA from 3T3 cells also is shown (lane 4) as verification that the COX-1 probe was reliable in an even less mRNA-enriched sample. These results are representative of identical results from three separate experiments and demonstrate the absence of COX-1 mRNA under basal (0.2% FBS) or stimulated (10% FBS) conditions (A). In contrast, COX-2 mRNA was readily detectable under basal and stimulated conditions (B). The probe used for Northern analysis readily detected COX-1 mRNA under basal conditions in 3T3 cells. COX/β-actin mRNA ratios for lanes 1–7 were 0.02, 0.09, 0.12, 3.62, 0.78, 0.74, and 1.46, respectively.