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. 2009 Jun 18;4(6):e5955. doi: 10.1371/journal.pone.0005955

Figure 1. UCH L1 is physically associated with β-catenin.

Figure 1

A. Endogenous β-catenin or UCH L1 were immunoprecipitated from KR4 (left) and 293 (right) cells. IPs were resolved in 10–12% PAGE and probed with indicated antibodies. Mouse and rabbit normal immunoglobulins were used as controls for IPs. B. Left panel: nuclear co-localization of UCH L1 and β-catenin. 293 cells were fixed in 4% PFA and double-immunostained with UCH L1 and β-catenin antibodies and red and green fluorescent secondary antibodies. Right panel: 293 cells were transfected with wild type HA-UCH L1 and myc-β-catenin expression vectors. After 24 h cells were fixed and probed with HA and myc antibodies. C. β-catenin is associated with wild type UCH L1, but not with inactive UCH L1 mutants. 293 cells were transfected with wild type and HA-UCH L1 mutants C90S and H161D, and UCH L1 was immunoprecipitated with HA antibody 48 h after transfection. IPs were resolved in 12% PAGE, transferred to PVDF membrane and probed with β-catenin antibody. Enzymatic activity of UCH L1 mutants was verified by in vitro hydrolysis of Ub-AMC (right panel).