Figure 3.

UbcH7 deficiency phenocopies ITCH phosphorylation. A, UbcH7^−/− HEK 293T cells were transiently transfected with HA-ubiquitin, NTAP-RIPK2, FLAG-ITCH, FLAG-ITCH S687A, Myc-UbcH7, or Myc-UBCH7 C86A as indicated. NTAP was precipitated under stringent washing conditions (1% SDS, 2 m NaCl). Western blotting was then performed with the indicated antibodies. Samples were subjected to Western blot analysis as indicated. Results shown are representative of three independent experiments. B, negative control and UbcH7^−/− HEK 293T cells were stimulated with 10 ng/ml TNFα for the indicated time points. Lysates were standardized for total protein and analyzed and subjected to SDS-PAGE and Western blot analysis.