1. A TPR Motif Cofactor Contributes to p300 Activity in the p53 Response
Constantinos Demonacos, Marija Krstic-Demonacos, Nicholas B La Thangue 
Molecular Cell 
Volume 8, Issue 1, Pages (July 2001)
DOI: /S (01)

2. Figure 1 Strap Is a TPR Motif-Containing Protein
(A) The primary amino acid sequence of Strap (440 amino acid residues). Residues within the six TPR motifs are set in bold.
(B) Diagrammatic summary of the distribution of the six TPR motifs in Strap (each TPR motif is indicated as I to VI).
(C) Alignment of the sequence from the six TPR motifs in Strap. The eight consensus residues derived from the alignment are shown below. The residue number is indicated at each end of the TPR motif. The consensus TPR motif is taken from Blatch and Lässle (1999).
(D) Expression profile of Strap determined by Northern blot analysis of RNA prepared from mouse tissues. As a control, the level of β-actin RNA was previously documented (Shikama et al., 1999)
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )

3. Figure 2 Strap Interacts with p300 and JMY in Mammalian Cells
(A) Two-hybrid assay in mammalian cells: the indicated expression vectors, either pVP16-JMY (0.5 μg) or pG4-Strap together with the control, pVP16 (0.5 μg), or G4 (0.1 μg) vectors were introduced into U2OS cells as indicated, together with the reporter pG5-luc (0.5 μg) and internal control pCMV-βgal (1 μg). The data represent the relative activity of luciferase to β-galactosidase and are the average values derived from two or more independent experiments.
(B) Two-hybrid assay was performed in U2OS cells as described in (A) using the indicated vectors, namely pVP16-Strap (0.5 μg) or pG4-p300 (0.5 μg). The data represent the average values derived from two or more independent experiments.
(C) Coimmunoprecipitation of JMY and Strap from U2OS cells transfected with pCMV-JMY (10 μg; lane 3) and/or pHA-Strap (10 μg; lanes 2 and 3) as indicated. pcDNA3 was cotransfected to equalize the total amount of transfected DNA.
(D) Coimmunoprecipitation of p300 and Strap from U2OS cells transfected with pCMV-p300 (30 μg; lane 3) and/or pHA-Strap (10 μg; lanes 2 and 3) as described in (A).
(E) Immunoblotting with the anti-Strap peptide antiserum performed on purified his-Strap (100 ng) with either immune (lanes 1 and 2) or preimmune (lane 3) in the presence of the Strap (lane 2) or control (lane 1) peptide.
(F) Immunoblotting with the anti-Strap peptide antiserum (lanes 2–4) performed on extracts prepared from U20S cells transfected with pCMV-HA-Strap in the presence of the Strap (lane 3) or control flag (lane 4) peptide. Lane 1 was immunoblotted with the anti-HA monoclonal antibody.
(G) Immunoblotting with the anti-Strap peptide antiserum performed on nontransfected A31 cell extracts with either preimmune (lane 1) or immune (lanes 2 and 3) serum in the presence of Strap peptide (lane 3).
(H) Immunoprecipitation from nontransfected A31 extracts was performed with anti-p300 Ab1 (lane 2) or control anti-HA (lane 3) antiserum followed by immunoblotting with the anti-Strap peptide antiserum. Lane 1 shows the input A31 extract
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )

4. Figure 3 Distinct Domains in Strap for Binding to p300 and JMY
(A) The indicated regions of Strap were in vitro translated and thereafter the translated material (about 10% of the input; lanes 1 and 4) was incubated with wild-type his-tagged JMY (about 1 μg; lane 3) or flag-tagged p300 (about 1 μg; lane 6) as described. In the control, either beads alone (lane 2) or beads incubated in Sf9 extract (lane 5) were used. The results are summarized in (D).
(B) The indicated regions of JMY were in vitro translated and thereafter the translated material (about 10% of the input; lane 1) was incubated with his-tagged Strap (about 1 μg; lane 3). The control treatment was as described in (A), and the results are summarized in (D).
(C) The indicated regions of p300 were either expressed as flag-tagged wild-type p300 or GST-p300 fusion proteins (1–595, 744–1571, or 1572–2370; about 1 μg) and incubated with in vitro-translated Strap as indicated. In the lower of the top two panels, binding of in vitro-translated Strap to p3001–595, p300744–1571, or p –2370 (lanes 3, 4, and 5) is shown, where the input (lane 1) and control bead activity (lane 2) is indicated. In the lower panels, in vitro-translated p300619–1303, p –2284, or p –1921 (about 10% of the input; lane 1) was assessed for binding to wild-type his-tagged Strap (about 1 μg; lane 3). The control treatment was as described previously, and the results are summarized in (D).
(D) Summary of the binding domains in Strap, JMY, and p300. The binding domains in JMY for p300, and in p300 for JMY, are taken from Shikama et al., 1999
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )

5. Figure 4 Strap Augments Formation of the p300/JMY Complex
(A) Levels of Strap, JMY, and p300 in U2OS cells after transfection with pHA-Strap (10 μg), pCMV-JMY (10 μg), or pCMV-p300 (10 μg) as indicated. The level of JMY increased 2.5-fold in lane 3 relative to lane 1.
(B) Coimmunoprecipitation of p300 and JMY from U2OS cells transfected with pHA-Strap (10 μg), pCMV-JMY (10 μg), or pCMV-p300 (10 μg) as indicated. The level of coimmunoprecipitated JMY was 5-fold greater in lane 3 relative to lane 2.
(C) Two-hybrid assay performed in U2OS cells with pG4-p300 (50 ng) and pVP-16-JMY (250 ng) in the presence of increasing amounts of pHA-Strap (0.5, 1.0, 3.0, and 5.0 μg), together with the reporter pG5-luc (1 μg) and internal control pCMV-βgal (1 μg).
(D) Biochemical binding assay using purified proteins in which flag-tagged p300 (0.5 μg) was incubated with his-tagged JMY (0.5 μg) in the presence of increasing levels of either Strap or Strap8–123 (1 and 2 μg) as indicated. The upper image represents a shorter exposure of the lower image, which is an extended exposure of the same gel
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )

6. Figure 5 Strap Augments the p53 Response
(A) U2OS cells were transfected with an increasing amount of pHA-Strap (5, 10, and 15 μg) as indicated and immunoblotted with either anti-p53 (DO1) monoclonal antibody (upper) or anti-HA (Y11) monoclonal antibody (lower).
(B) Early passage p53−/− MEFs were transfected with the expression vectors for p53 (5 μg) together with an increasing amount of pHA-Strap (5, 10, and 15 μg) as indicated. Cell extracts were prepared and immunoblotted as described in (A).
(C) U2OS cells were transfected with either pcDNA3, pHA-Strap (15 μg), pCMV-JMY (10 μg), or pCMV-p300 (5 μg), as indicated. Transfected cells were treated with cyclohexamide (10 μg/ml) and at the indicated time points, extracts were prepared and immunoblotted to detect the levels of p53.
(D) Early passage p53−/− MEFs were transfected with expression vectors for wild-type p53 (5 μg) together with hDM2 (20 μg) and Strap (15 μg) as indicated. Cell extracts were prepared and immunoblotted as described in (A).
(E) The indicated p53 reporter constructs, pWWP-luc, pBax-luc, and pTG13 (1 μg), together with expression vectors for p53 (0.025 μg) and Strap (2 μg and 5 μg) were transfected into SAOS2 cells as indicated.
(F and G) SAOS2 cells were transfected with expression vectors for wild-type p53 or p5322/23 (4 μg) in the presence or absence of Strap (7 μg) together with pCMV CD20 (7 μg), as indicated. The % change in the size of the sub-G1, G1, S, and G2/M population is shown. In (F and G), p53 caused about 37% of the transfected cells to enter apoptosis, compared to 17% with the vector alone.
(H) SAOS2 cells were transfected with expression vectors for wild-type p53 (2 μg) in the presence of Strap or Strap8–123 (8 and 12 μg), or cotransfected together (8 μg Strap8–123 and 8, 10, or 12 μg Strap) as indicated. Cells were harvested at 48 hr and immunoblotted for p53 or Strap levels using anti-p53 (DO-1) or anti-HA, respectively.
(I) SAOS2 cells were transfected with pBax-luc (1 μg) together with expression vectors for p53 (100 ng) and Strap8–123 (6 μg) as indicated.
(J) U2OS cells were transfected with pBax-luc (1 μg) together with expression vectors for Strap or Strap8–123 (3 and 6 μg), or cotransfected together (6 μg Strap8–123 with 3 and 6 μg of Strap) as indicated.
(K) U2OS cells were transfected with expression vectors for Strap (8 and 12 μg) or Strap8–123 (20 and 30 μg), or cotransfected together (20 μg Strap8–123 with 8 and 12 μg of Strap) as indicated. Cells were harvested at 48 hr and immunoblotted for p53 or Strap levels using anti-p53 (DO-1) or anti-HA, respectively
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )

7. Figure 6 Strap Is a Stress-Responsive Protein
(A) p53−/−/mdm2−/− MEFs were transfected with either pG4-p53 (25 ng), pHA-Strap (4 μg), or pCMV-HDM2 (3 μg) together with pG5-luc (1 μg).
(B) p53−/−/mdm2−/− MEFs were transfected with expression vectors for wild-type p53 (25 μg), Strap (2.5 μg), or hDM2 (1.5 μg) together with pBax-luc (1 μg) and the internal control pCMV-βgal (1 μg).
(C) U2OS cells were transfected with either pHA-Strap (5 μg), pCMV-JMY (5 μg), or pCMV-NAP2 (5 μg) and treated as described either with or without etoposide (200 or 400 nM). The levels of exogenous Strap, JMY, and NAP, and endogenous p53, were determined by immunoblotting with the relevant antibodies, quantitated by phosphoimaging, and thereafter presented graphically. The level of protein detected in the nontreated cell was given an arbitrary value of 1.0. •, p53; □, Strap; ■, JMY; ○, NAP.
(D) Levels of JMY in U2OS cells after transfection with pCMV-JMY (5 μg) and pCMV-p300 (5 μg), together with pHA-Strap (5 μg) and etoposide treatment (200 nM) as indicated.
(E) Coimmunoprecipitation of p300 and JMY from the cell extracts described in (D).
(F) Two-hybrid assay performed in U2OS cells with pG4-p300 (100 ng) and pVP16-JMY (1 μg) in the presence of pHA-Strap (2.5 μg) and etoposide (200 nM) as indicated, and the reporter pG5-luc (1 μg) and internal control pCMV-βgal (1 μg). The data represent the relative activity of luciferase to β-galactosidase, and are the average values derived from two different readings
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )

8. Figure 7 Characterization of Endogenous Strap
(A) U2OS cells were transfected with the expression vector encoding Strap (5 μg) and treated as described with etoposide (200 nM). Extracts were prepared and immunoblotted with the anti-p53 monoclonal antibody DO-1.
(B) Immunoprecipitation from the extracts described in (A) with anti-HA monoclonal antibody by immunoblotting with anti-p53 rabbit polyclonal antibody.
(C) A31 cells were treated as indicated with etoposide (panel i), ultraviolet light (UV; panel ii), or ionizing radiation (IR; panel iii) and extracts were prepared followed by immunoblotting with anti-Strap peptide antiserum. In UV treatment, the time of harvesting after treatment (in hours) is indicated. The Strap polypeptide is indicated; a minor Strap-related polypeptide is seen in panels ii and iii.
(D) Immunoprecipitation from U2OS cells was performed from nontransfected extracts prepared from either normal or etoposide-treated (400 nM) cells with the anti-p300 monoclonal antibody (Ab1) followed by immunoblotting with either anti-p300 (top), anti-JMY (middle), or anti-Strap (lower) peptide antibodies. The level of p300, JMY, and Strap in the cell extracts is shown. Quantitation of each immunoprecipitated polypeptide indicated that while p300 levels were similar, JMY and Strap were increased by about 100% and 70%, respectively in the stressed relative to nonstressed cell extracts. Anti-HA monoclonal antibody was used for the control immunoprecipitation (lanes 5 and 6).
(E) Model for role of Strap in the p53 response: Strap is induced by stress, and activates p300 coactivator function and blocks MDM2 effects. As a result, the p53 response is triggered. Stress-activated phosphokinases (including ATM, ATR, and chk1/2) act upon p53 and MDM2 to alter p53 stability and enhance the interaction of p53 with the p300 coactivator complex. Strap induction by stress (“?” indicates that it is not known whether the same phosphokinases are involved) augments the activity of the p300 coactivator complex
Molecular Cell 2001 8, 71-84DOI: ( /S (01) )