JAZ associates with p53 in various mammalian cell lines and directly binds p53 in vitro independent of dsRNA. (A-B) FLAG-JAZ in pcDNA3 (Invitrogen) was transfected into COS-7, CV-1, or p53-null SAOS-2 cells with or without p53wt/pCMV (BD CLONTECH). After 24 or 48 hours, cells were lysed in the lysis buffer (10 mM Hepes [pH 7.3], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail σ). Coimmunoprecipitation (IP) followed by immunoblotting (IB) was performed using antibodies against FLAG and p53 (DO-1 or FL393). V indicates pcDNA3; F-J, FLAG-JAZ/pcDNA3; p53, p53wt/pCMV; and 20% or 50% input, 50 μg cell lysate. (C) The rabbit JAZ antibody (JAZ111 and the control antibody as described in “Materials and methods”) was used in IP and IB endogenous JAZ from 1- or 0.5-mg whole-cell lysate from NIH3T3, NFS/N1.H7, or p53-deficient M1 cells, and JAZ-associated p53 was immunoblotted using a p53 antibody (PAb 246). To facilitate detection of endogenous JAZ or p53 protein, H7 or M1 cells were deprived of IL-3/serum for 2 hours to increase JAZ or p53 expression (as shown in Figure 1). “10% input” indicates 100 or 50 μg cell lysate; +, JAZ111; and –, the control Ab. (D) Subcellular fractionation of p53^+/+ MEFs and CV-1 cells was performed as described in “Materials and methods.” The JAZ111 antibody was used to coimmunoprecipitate endogenous JAZ from 1 mg lysate of the nuclear or cytoplasmic fraction, followed by IB using JAZ111 and antibodies against p53 and PCNA (a nuclear protein as a control). Nuc indicates nuclear fraction; Cyt, cytoplasmic fraction; and 10% Input, 100 μg nuclear or cytoplasmic lysate. (E) GST and GST-JAZ glutathione-Sepharose beads were incubated with 100 ng purified, recombinant p53 in the in vitro binding buffer followed by IB using DO-1 (top panel). The purified proteins were treated by RNase V1 as described in “Materials and methods.” The bottom panel represents Fast-green staining of the blot to show expression of GST or GST-JAZ. rp53 indicates recombinant p53; 20% Input, 20 ng rp53. Densitometric quantitation of bound rp53 versus GST-JAZ was performed following treatment with or without RNase V1. The average ratio of p53/JAZ from 3 independent experiments is shown (the ratio is set to 1.00 for the control/no treatment sample). (F) The GST-JAZ NF beads containing the mutant in which all 4 zinc fingers are point mutated by changing C₂H₂→C₂AH³²  were also used to pull down rp53.