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

FIGURE 3.

FIGURE 3.

p11 is polyubiquitinated in the absence of A2. A, HEK 293 cells were transiently transfected with FLAG-p11 for 48 h. The cells were pretreated with cycloheximide (CHX) (10 μg/ml, 30 min) and then incubated with or without MG132 (10 μm) as indicated. Lysates were immunoblotted for FLAG-p11, A2, and GAPDH. B, HEK 293 cells were cotransfected with plasmids encoding FLAG-p11 and HA-ubiquitin (HA-Ub). Cell lysates (500 μg) were either immunoprecipitated (IP) with anti-HA and immunoblotted (WB) with anti-FLAG IgG (left panel) or immunoprecipitated with anti-FLAG and blotted with anti-HA IgG (right panel). C, dominant negative ubiquitin mutants, K48R and K29R, or empty control vectors were cotransfected with FLAG-p11, and cell lysates were subjected to immunoblotting with anti-p11 and anti-actin IgG after 36 h. D, FLAG-p11 was transfected with either HA-wt- or HA-K48R-ubiquitin into HEK 293 cells. Cell lysates were coimmunoprecipitated with anti-FLAG IgG and either probed with anti-HA antibody (top panel) or immunoblotted with anti-p11 (lower panel).