Figure 3. INZ treatment enhances the interaction of MDM2 with L5 and L11 by inducing ribosome-free form of RPL5 and RPL11.
(A) H460 cells were treated with 2 μM INZ or INZ (O) for 18 hr. Cell lysates were used for IP with anti-MDM2 antibodies or anti-L11 antibodies followed by IB using anti-RPL5, RPL11 or MDM2 antibodies. (B and C) HCT116 cells were transfected with siRNAs against RPL5 and RPL11, or control, for 48–72 hr and treated with 2 µM INZ 18 hr before harvesting, followed by IB using indicated antibodies or subG1 analysis by flow cytometry. (D) Ribosomal profile assay. Cytoplasmic extracts containing ribosomes from H460 cells treated with or without 2 µM INZ for 18 hr were subjected to a 10–50% linear sucrose gradient sedimentation centrifugation. Fractions were collected and subjected to IB with anti-RPL11, anti-RPL5, anti-p53, or anti-MDM2 antibodies. The distribution of ribosomes is indicated.