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. 2018 May 29;92(12):e02034-17. doi: 10.1128/JVI.02034-17

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Copyright © 2018 Chalabi Hagkarim et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

PMC Copyright notice

FIG 9 — Adenovirus early region E1B55K interacts with Tab182 in vitro and in vivo. (A and B) Ad12E1HER2 (A) and Ad5E1HEK293 (B) cell lysates containing 500 μg total protein were incubated with 5 μg either GST-Tab182 or GST-PRMT1 or with GST alone. Protein complexes were captured by glutathione-agarose beads and subjected to SDS-PAGE and Western blotting (WB) with the antibodies indicated. (C and D) Ad5E1HEK293 (C) and Ad12E1HER2 (D) cell lysates (500 μg total protein) were incubated with antibodies against Tab182 and collagen IV together with IgG (nonspecific binding controls). Immunocomplexes were isolated by using protein G-agarose beads and subsequently resolved by SDS-PAGE and Western blotting using antibodies against Ad5E1B55K/Ad12E1B55K proteins. IP, immunoprecipitation. (E) GFP-Tab182 was transfected into Ad5E1HEK293 and Ad12E1HER2 cell lines, which were harvested after 48 h. Cell lysates (500 μg total protein) were incubated with Ad5E1B55K and Ad12E1B55K antibodies together with IgG. Western blotting was performed with an antibody against Tab182. (F) HeLa cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 μg total protein) were immunoprecipitated with an antibody against Tab182 or rabbit IgG. Western blotting was performed with an antibody against HA. (G) Overexposed version of a portion of the Western blot shown in panel F. (H) Ad5E1HEK293 cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 μg total protein) were immunoprecipitated with an antibody against HA or mouse IgG. Western blotting was performed with an antibody against p53. (I) HEK293FT cell lysates (500 μg protein) were incubated with antibodies against Tab182, collagen IV, or the IgG control. Western blotting was performed with an antibody against SV40T antigen. (J and K) Ad5E1HEK293 (J) and Ad12E1HER2 (K) cell lysates (500 μg total protein) were incubated with antibodies against CNOT1 and collagen IV together with IgG. Western blotting was performed with antibodies against Ad5E1B55K and Ad12E1B55K proteins. In all cases, the whole-cell lysates contained 15 μg of protein. Although only limited areas of the Western blots are shown, no additional bands were seen in the original autoradiographs.

FIG 9 — Adenovirus early region E1B55K interacts with Tab182 in vitro and in vivo. (A and B) Ad12E1HER2 (A) and Ad5E1HEK293 (B) cell lysates containing 500 μg total protein were incubated with 5 μg either GST-Tab182 or GST-PRMT1 or with GST alone. Protein complexes were captured by glutathione-agarose beads and subjected to SDS-PAGE and Western blotting (WB) with the antibodies indicated. (C and D) Ad5E1HEK293 (C) and Ad12E1HER2 (D) cell lysates (500 μg total protein) were incubated with antibodies against Tab182 and collagen IV together with IgG (nonspecific binding controls). Immunocomplexes were isolated by using protein G-agarose beads and subsequently resolved by SDS-PAGE and Western blotting using antibodies against Ad5E1B55K/Ad12E1B55K proteins. IP, immunoprecipitation. (E) GFP-Tab182 was transfected into Ad5E1HEK293 and Ad12E1HER2 cell lines, which were harvested after 48 h. Cell lysates (500 μg total protein) were incubated with Ad5E1B55K and Ad12E1B55K antibodies together with IgG. Western blotting was performed with an antibody against Tab182. (F) HeLa cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 μg total protein) were immunoprecipitated with an antibody against Tab182 or rabbit IgG. Western blotting was performed with an antibody against HA. (G) Overexposed version of a portion of the Western blot shown in panel F. (H) Ad5E1HEK293 cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 μg total protein) were immunoprecipitated with an antibody against HA or mouse IgG. Western blotting was performed with an antibody against p53. (I) HEK293FT cell lysates (500 μg protein) were incubated with antibodies against Tab182, collagen IV, or the IgG control. Western blotting was performed with an antibody against SV40T antigen. (J and K) Ad5E1HEK293 (J) and Ad12E1HER2 (K) cell lysates (500 μg total protein) were incubated with antibodies against CNOT1 and collagen IV together with IgG. Western blotting was performed with antibodies against Ad5E1B55K and Ad12E1B55K proteins. In all cases, the whole-cell lysates contained 15 μg of protein. Although only limited areas of the Western blots are shown, no additional bands were seen in the original autoradiographs.