Figure 7

SAMD12-AS1 promotes HDM2-mediated ubiquitination of p53. (a) L02 cells were cotransfected with pCMV Myc-HDM2 and control plasmid or SAMD12-AS1-expressing plasmids. Cell lysates were subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies (left). The amount of p53 bound to Myc-HDM2 was quantified (right). (b,c) L02 cells were transfected with control, SAMD12-AS1, SAMD12-AS1(1-350) or SAMD12-AS1(351-701) plasmids. Cell lysates were subjected to immunoprecipitation with anti-p53 antibody (b, left) or anti-HDM2 antibody (c, left), followed by immunoblotting with antibodies against HDM2, p53 or NPM1 as indicated. The relative amount of HDM2 bound to p53 and NPM1 bound to HDM2 were quantified (b,c, right). (d) GST-HDM2 and His-NPM1 were mixed with in vitro transcribed SAMD12-AS1, SAMD12-AS1(1-350) or SAMD12-AS1(351-701) and then subjected to pulldown with glutathione beads followed by immunoblotting with anti-GST and anti-His antibodies (left). The amount of His-NPM1 bound with GST-HDM2 was quantified (right). (e) L02 cells were cotransfected with pCMV His-Ub and control plasmid or SAMD12-AS1, SAMD12-AS1(1-350) or SAMD12-AS1(351-701)expression plasmids. Then, cells were treated with MG132 for 6 h, and cell lysates were subjected to His pulldown and immunoblotted with anti-p53 antibody (upper panel). The relative amount of ubiquitinated p53 (Ub-p53 in short) was quantified (lower panel). The relative amounts of p53, HDM2, NPM1, His-NPM1 and Ub-p53 were quantified using ImageJ. **P < 0.01; ***P < 0.001; ns, not significant. The data are representative of three independent experiments.