Fig. 7

RF-Id inhibits XIAP-cIAP2 interaction and NFκB activation. Total protein extracts were subjected to immunoprecipitation with 2 μg of anti-XIAP or anti-cIAP2 for 24 h at 4 °C. Immune complexes were collected with 50 μl of protein A-agarose for 16 h at 4 °C. The protein A-agarose/immune complex was washed twice with cold PBS, resuspended in 20 μl of SDS-loading buffer, heated to 95 °C for 5 min and used for Western blotting analysis using anti-XIAP or anti-CIAP2 . Representation of the c-IAP2 and XIAP complexes was expressed as the mean of the ratio between the relative intensities of the bands associated with the c-IAP2/XIAP complexes versus the bands associated with total C-IAP2 and XIAP, respectively. The intensities of the bands were expressed as arbitrary units when compared to that of the untreated cells. Error bars showed standard deviation from the mean in at least three independent experiments. Bars, SDs.** p ≤ 0.01 (a). U87MG cells were treated for 72 h with IC:50 of RF-Id or 500nM Bortezomib for 24 h or a combination of RF-Id and Bortezomib. Cell lysates were incubated with anti-human c-IAP2, pIKKα/β, IKKα/β, pIKBα, IKBα and NFκB antibodies and analysed by Western Blotting; the housekeeping protein α-tubulin was used as loading control. The experiments were repeated three times giving always similar results. Quantitation of the bands for each proteins are reported in Additional file 1: Figure S4 (b)