Effects of RNAi-mediated knock-down on the shedding of RAGE.

PAC1/RAGE cells were transfected with stealth RNAi oligonucleotide duplexes (Invitrogen) targeting either ADAM10 (AD10), ADAM17 (AD17), MMP9 or MMP2. As control (-) cells were transfected with a stealth RNAi control oligonucleotide duplex (Invitrogen). Experiments were performed 48 h after transfection. Secretion of RAGE was analyzed under unstimulated (H₂O, n = 10) and PACAP-induced (300 nM, n = 5) conditions for 3 h. (A) Typical Western blots and gelatin zymography are shown, (B) Quantitative analysis of secreted RAGE. RAGE secreted into the cell culture supernatant and full-length RAGE in cell lysates were detected with antibody Mab5328. The enzymatic activity of MMP9 and MMP2 in cell culture supernatants was determined by gelatin zymography as described in Materials and Methods. In cell lysates full-length RAGE (fl-RAGE), ADAM10 (pro and mature (m) forms), and ADAM17 (pro and mature (m) forms) were detected with suitable antibodies (see “Materials and Methods”). As a loading control actin was detected in cell lysates by Western blotting. Shown in the quantitative analysis (B) are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (ns  =  P>0.05; *  =  P<0.05; **  =  P<0.01; ***  =  P<0.001).