p63 and p73 are required for p53-dependent apoptosis in response to DNA damage

✍ Scribed by Flores, Elsa R.; Tsai, Kenneth Y.; Crowley, Denise; Sengupta, Shomit; Yang, Annie; McKeon, Frank; Jacks, Tyler

• Book ID: 109803299
• Publisher: Nature Publishing Group
• Year: 2002
• Tongue: English
• Weight: 537 KB
• Volume: 416
• Category: Article
• ISSN: 0028-0836
• DOI: 10.1038/416560a

✦ Synopsis

experiments shown in Fig. , b were exactly as described 5 , except that the binding and washing buffer for the GST -LHP1 results shown in Fig. consisted of 50 mM sodium phosphate and 25 mM NaCl at pH 6.0. For Fig. , bound proteins were separated by 4 -20% gradient SDS -PAGE, blotted, and detected with an anti-GST monoclonal antibody (Pierce). For Fig. , the bound biotinylated peptides (Upstate Biotechnology) were separated by 18% SDS -PAGE, blotted, and detected with streptavidin HRP conjugate (Upstate Biotechnology).
Interaction of CMT3 with LHP1
A six-histidine fusion construct was made by cloning full-length LHP1 into the XhoI and PstI sites of pRSETB, and expressed in E. coli strain BL21. Proteins were purified on Ni-NTA-agarose (Qiagen) and eluted with 100 mM imidazole before mixing with glutathione-agarose-bound GST fusion proteins. Binding and wash buffers were the same as in the peptide binding assays 5 . Bound proteins were separated by 4-20% gradient SDS -PAGE, blotted, and detected with INDIA HisProbe-HRP (Pierce).