Fig. 7.

A_2B adenosine receptor (A_2B AR) knockdown in Caco2-BBE (C2) cells using siRNA. A: C2 cells were untransfected (UT) or transfected with a negative control siRNA (NC siRNA) or a specific A_2B AR siRNA (A_2B AR siRNA), and then total RNA was isolated 72 h later for real-time PCR analysis. Values are means ± SE of 5 independent experiments each of which was done in triplicate. Relative A_2B AR mRNA expression level is expressed as a percentage of UT normalized to GAPDH. siRNA knockdown of the A_2B AR significantly reduced its mRNA expression level (*P < 0.001 for A_2B AR siRNA compared with UT and NC siRNA, by ANOVA). B: a representative Western blot analysis of A_2B AR total protein expression. A_2B AR protein expression was evaluated in C2 cell lysate (30 µg protein/lane; NC, C2 cells transfected with the negative control siRNA; A2B, C2 cells transfected with the siRNA targeting the A_2B AR). The lower half of the same blot was probed with an anti-β-actin antibody to normalize loading of protein in each lane (bottom). C: densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 4 independent experiments of relative A_2B AR abundance to β-actin and are presented as a percentage of the UT value. siRNA knockdown of A_2B AR significantly reduced A_2B AR total protein expression (*P < 0.001 and < 0.01 for A_2B AR siRNA compared with UT and NC siRNA, respectively, by ANOVA).