Results: 5

1.
Figure 3

Figure 3. From: Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

Minimal association of E7ER with pRb and induction of p107 association on E2 treatment. Confluent starved cultures were treated overnight with E2 (1 μM) (+) or ethanol (−) and immunoprecipitated with anti-ER antibodies (A and C) and then sequentially with anti-pRb antibodies (B). Western blots were probed with anti-Rb antibodies (A and B) or anti-p107 antibodies (C). Each lane consists of lysate from 2 × 107 cells expressing the E7ER construct (3-3 and 1-1) or the ER construct (ER-3).

K. Smith-McCune, et al. Proc Natl Acad Sci U S A. 1999 June 8;96(12):6999-7004.
2.
Figure 5

Figure 5. From: Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

Immunofluorescent localization of E7ER. (AD) Altered intracellular localization on E2 treatment. Serum-starved confluent cultures of C7/E7ER 1-1 cells (A and B) or C7/ER-3 (C and D) were treated overnight with 1 μM E2 (B and D) or ethanol (A and C) and prepared for immunofluorescence as described. (Bar = 20 μm.) (EJ) In situ lysis reveals differential nuclear localization of E7ER on E2 treatment. Serum-starved cultures of C7/E7ER 1-1 cells were treated overnight with 1 μM E2 (F, H, and J) or ethanol alone (E, G, and I). Cells were lysed in hypotonic buffer before fixation and then prepared for immunofluorescence with anti-ER antibodies (E and F), DAPI (G and H) or rabbit IgG (I and J). (Bar = 50 μm.)

K. Smith-McCune, et al. Proc Natl Acad Sci U S A. 1999 June 8;96(12):6999-7004.
3.
Figure 2

Figure 2. From: Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

E7 and E7ER interactions with pRb in a mammalian two-hybrid system. Cells were transfected with the luciferase reporter (g5E1Blux), the CAT reporter, and various constructs as indicated. Luciferase values were normalized to transfection efficiency as described in the text. Fold induction refers to the normalized luciferase value of the experimental cells (average of two plates) divided by the normalized luciferase value of cells transfected with the luciferase reporter and CAT reporter alone. E2 (1 μM), tamoxifen (1 μM), or ethanol was added as indicated. PVU1 cells were used in A, whereas CV1 cells were used in B and C; the enhanced induction of luciferase activity in A may be because of the fact that the large tumor antigen expressed by these cells replicates the Gal4-based plasmids that have an SV40 origin. Columns represent the average and error bars represent the SE of duplicate transfections. See Materials and Methods for descriptions of g5E1BLux, g4Rbp, and g4Rbpm.

K. Smith-McCune, et al. Proc Natl Acad Sci U S A. 1999 June 8;96(12):6999-7004.
4.
Figure 4

Figure 4. From: Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

E7ER preferentially binds to p130. Experiments were performed as in Fig. 3. In A, − or + refers to the absence or presence of E2. In C and D, untreated cell lysates were immunoprecipitated first with anti-ER antibodies and then sequentially with anti-p130 antibodies; immunoprecipitates were run in neighboring lanes to allow comparison of the proportion of protein by densitometric scanning of the autoradiograms (E). E7 mutER is a clonal population expressing a point mutation in the Rb-binding pocket (Gly-24). A second clonal cell line gave identical results. Data in E represents comparisons by densitometry scanning of the amounts of pRb, p107, and p130 immunoprecipitated with anti-ER antibodies compared with the sum of the amounts immunoprecipated with anti-ER and subsequently with the relevant Rb-family member antibody. Data from two (p107) or three (pRb and p130) independent experiments were averaged; error bars represent the SEM.

K. Smith-McCune, et al. Proc Natl Acad Sci U S A. 1999 June 8;96(12):6999-7004.
5.
Figure 1

Figure 1. From: Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.

Structure and function of the E7ER chimera. (A) pMXE7ER contains the E7ER chimera in a retroviral vector with the neomycin-resistance gene. X, Xho; B, BamHI; E, EcoRI; C, ClaI. (B) Lysates of 3 × 105 cells transfected with the ER construct (C7/ER-3) and E7ER (C7/E7ER 3-3) were analyzed by using Western blot analysis with anti-ER rabbit antiserum (S. Robbins). Positions of 45- and 29-kDa molecular mass markers are shown to the right. (C) [3H]Thymidine incorporation was measured in starved E7ER-expressing cells (C7/E7ER 1-1) or a pool of cells transfected with vector alone (C7/neo) in response to increasing doses of E2. Each point is the average of duplicate wells; duplicate cpm values varied by <10%. (D) Induction of DNA synthesis was measured in response to 1 μM E2 in clonal cell populations expressing the ER domain (C7/ER3), wild-type E7 in the context of the ER chimera (E7ER 1-1 and 3-3), and mutant E7 alleles containing a point mutation in the Rb-binding pocket (E7*ER 4-1 and 4-3) or a deletion of the Rb-binding pocket (E7*ER 5-4). Bar graphs represent the mean from 4 (C7/ER3), 12 (E7ER 1-1), 19 (E7ER 3-3), 3 (E7*ER 4-1 and 4-3), or 2 (E7*ER 5-4) independent assays. Error bars represent the SEM. (E) Cells (104) were plated in soft agar, and the total number of colonies greater than 6–8 cells in size was counted after 2 (C7/E7, C7/E7ER 1-1 and 3-3) or 4 (C7/ER3) weeks. Results are expressed as a ratio of the total number of colonies in the presence as compared with the absence of E2.

K. Smith-McCune, et al. Proc Natl Acad Sci U S A. 1999 June 8;96(12):6999-7004.

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