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Figure 6


Fig. 6. UBPY and AMSH deubiquitylate VAMP8. (A) HeLa cells were transfected with FLAG-VAMP8, together with HA-tagged UBPY, UBPYC748A, AMSH or AMSHD348A. FLAG-VAMP8 was immunoprecipitated from their lysates with anti-FLAG antibody, and immunoblotted with FK2 (top), anti-HA (second from top) and anti-FLAG (third from top) antibodies. The expression levels of HA-tagged DUB constructs were assessed by immunoblotting of the total lysates with anti-HA antibody (bottom). (B) HeLa cells were transfected with FLAG-VAMP8, together with the mock, two UBPY siRNA or two AMSH siRNA expression vectors. FLAG-VAMP8 was immunoprecipitated from their lysates with anti-FLAG antibody, and immunoblotted with FK2 (top) and anti-FLAG (second from top) antibodies. The expression levels of UBPY and AMSH were assessed by immunoblotting of the total lysates with corresponding antibodies (third and fourth from top). As a loading control, the lysates were also blotted with anti-tubulin antibody (bottom).





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