Journal:

Article Title: Dimerization with PEBP2? protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation

doi: 10.1093/emboj/20.4.723

Figure Lengend Snippet: Fig. 6. Mutations that abrogate heterodimerization eliminate the ability of PEBP2β to protect RUNX1 from proteolysis. (A) Transient expression of RUNX1, PEBP2β2 and their derivatives alone or in combinations in P19 cells. Whole-cell extracts were prepared from half of the transfected cells and analyzed by western blotting using anti-HA and anti-β2. Notations above the gel pattern indicate: β2/AA, PEBP2β2(L64A, N104A); β2, PEBP2β2; G108R, RUNX1(G108R). (B) The remaining half of the transfected cells in (A) were lysed and immunoprecipitated with polyclonal anti-HA antibody and then analyzed by western blotting using monoclonal anti-HA and polyclonal anti-β2. The strong band present in all lanes in the upper part of the gel is non-specific. (C) RUNX1 or RUNX1(G108R) was transiently expressed in P19 cells with increasing amounts of PEBP2β2 (lanes 2–4) or PEBP2β2(L64A, N104A) (lanes 5–7). Where indicated, proteasome inhibitor MG132 was added to the cell culture medium 24 h after transfection. The notations above the panel are the same as in (A). Whole-cell extracts were prepared after 36 h of incubation and analyzed by western blotting using anti-αB1, anti-β2 and anti-luc (internal control).

Article Snippet: Chemicals and reagents Lysosomal protease inhibitor E64, calpain inhibitor ALLM, calpain and proteasome inhibitor ALLN, proteasome inhibitors MG132 and lactacystin were purchased from MBL (Nagoya, Japan).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Cell Culture, Incubation

Journal:

Article Title: Dimerization with PEBP2? protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation

doi: 10.1093/emboj/20.4.723

Figure Lengend Snippet: Fig. 5. Stabilization of RUNX1 by heterodimerization with PEBP2β. (A) Transient expression of RUNX1 (lanes 1–5) without (lane 1) or with increasing amounts (lanes 2–4) of PEBP2β. Lane 5 represents the cells treated with MG132. Whole-cell extracts of P19 cells were analyzed using anti-αB1, anti-β2 and anti-luc (an internal control). (B) Effects of exogenously expressed PEBP2β2 and proteasome inhibitor MG132 on the stability of endogenous RUNX1 in 32D cl3 cells. Whole-cell extracts were analyzed by using anti-αB1 and anti-β2. (C) Stabilization of RUNX2 and RUNX3 by PEBP2β. RUNX2 or RUNX3 with or without PEBP2β2 (shown as β2) was exogenously expressed in P19 cells and whole-cell extracts were analyzed by anti-Runt domain, anti-RD(10B7G8) and anti-β2.

Article Snippet: Chemicals and reagents Lysosomal protease inhibitor E64, calpain inhibitor ALLM, calpain and proteasome inhibitor ALLN, proteasome inhibitors MG132 and lactacystin were purchased from MBL (Nagoya, Japan).

Techniques: Expressing