Figure 6
LZAP binds both p53 and HDM2. (a) Immunoblots of LZAP and p53 of U2OS lysates following transfection with control or LZAP siRNA, and treatment with vehicle or MG132 for 4 h. (b) Immunoblots of LZAP, p53 and Wip1 of U2OS (parental and LZAP CRISPR) lysates following transfection with control or Wip1 siRNA. All lanes were run on the same gel; solid line indicates where images were cropped. (c) Immunoblots of LZAP, p53 and MDM2 of U2OS lysates following transfection with combinations of non-targeting siRNA, LZAP siRNA or one of two siRNAs targeting MDM2. (d) U2OS cells were transfected with indicated plasmids encoding tagged Flag-LZAP or GFP-p53. Immunoprecipitates were prepared using Flag affinity matrix to pulldown LZAP, resolved on SDS–polyacrylamide gel electrophoresis, and immunoblotted by antibodies recognizing Flag(-LZAP) or GFP(-p53). Expression of LZAP and p53 was confirmed by immunoblotting whole-cell lysates with Flag or GFP antibodies, respectively. (e) Purified LZAP was incubated with p53 along or together with GST-HDM2 or GST proteins followed by pulldown with agarose beads conjugated with LZAP or p53 antibodies and detection with HDM2, LZAP, p53 or GST antibodies (see Materials and methods section). (f) Purified p53 was incubated with GST-HDM2 along or together with LZAP in the presence or absence of nutlin. Similarly, LZAP was incubated with GST-HDM2 in the presence or absence of nutlin, followed by pulldown with agarose beads conjugated with LZAP or p53 antibodies and detection with HDM2, LZAP or p53 antibodies.
