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. 2008 Jul 11;283(28):19192–19200. doi: 10.1074/jbc.M800100200

FIGURE 4.

FIGURE 4.

The N-terminal tail domain of A2 protects p11 from ubiquitin-dependent proteolysis. A, HEK 293 cells were cotransfected with A2-myc and either FLAG-p11 or empty vector. Immune complexes were precipitated with anti-FLAG IgG and immunoblotted anti-Myc IgG (upper panel). The same blot was stripped and reprobed with anti-FLAG IgG (lower panel). B, HEK 293 cells were cotransfected with plasmids encoding FLAG-p11 and C-terminal Myc-tagged A2. After 48 h, cells were pretreated with cycloheximide (CHX) (10μg/ml, 30 min) and then incubated with or without MG132 (10 μm, 8 h). Lysates were immunoblotted with anti-FLAG, anti-A2, and anti-actin. C, HEK 293 cells were cotransfected with HA-ubiquitin, plus A2-myc and FLAG-p11 at molar ratios of 1:1, 2:1, 3:1, and 4:1. Lysates were either immunoprecipitated (IP) with anti-HA IgG and blotted with anti-FLAG IgG (upper panel) or immunoblotted (WB) with anti-Myc, anti-FLAG, and anti-actin IgG (lower panels). D, HEK 293 cells were transfected for 36 h with Myc-tagged full-length or N-terminal truncation mutants of A2, as well as FLAG-p11. Cells were treated with cycloheximide (10 μg/ml, 30 min) in the presence or absence of MG132 (10 μm, 8 h), and lysates were immunoblotted with anti-FLAG, anti-Myc, and anti-actin IgG.