Figure 2 Quantitative real‐time RT‐PCR and ELISA analysis of anti‐β₂GPI antibody‐mediated gene regulation in HUVEC. Cells were incubated with different anti‐β₂GPI antibody preparations (P1, P2, P4, P5, 50 μg/ml) or with control normal IgG (N3, N4, 50 μg/ml) or, TNFα for 4 h and total RNA isolated and processed for real‐time PCR analysis. Antibody preparations N3, N4, P1, P2 and P4 were previously used in the microarray experiments. Gene expression levels were normalised to the β‐actin mRNA level. The results show change in expression level relative to control normal IgG (N3) level and represent the mean of duplicate samples from two independent experiments. (A) shows data for SOD2, CX3CL1 and E‐selectin. TNFα induced high‐level expression of these genes but induction levels were off the scale and omitted from the figure, (B) shows data for CSF, FGF, Tenascin C (TNC) and ID2. TNFα‐regulated changes in levels of expression are included for comparison. The effect of four control normal IgG and four APS‐derived anti‐β₂GPI antibody preparations (used in microarray experiments) on E‐selectin expression (C) and IL‐8 secretion (D) was determined by ELISA. Antibodies were incubated with the cells for 4 h at 50 μg/ml. Results show mean ±SEM of triplicate samples from a representative experiment (one of three). * = p<0.03 as determined by two‐tailed Mann–Whitney U Test. TNFα also induced high levels of E‐selectin and IL‐8 in HUVEC as measured by ELISA (data not shown).