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. 2017 Dec 6;293(3):1100–1105. doi: 10.1074/jbc.RA117.000408

Figure 2.

Figure 2.

Phe-63 mediates the phosphorylation-dependent coupling between UbcH7 and ITCH. A, HEK 293T cells were transduced with lentivirus containing CRISPR-Cas9 with either a negative control-RNA guide or UbcH7 Guide 1-RNA. Lysate from pooled select UbcH7−/− clones was run next to controls on SDS-PAGE and probed for UbcH7 and GAPDH as shown. B, negative control and UbcH7−/− HEK 293T cells were transiently transfected with HA-ubiquitin, NTAP-RIPK2, and ITCH or ITCH C830A to assess ITCH ubiquitination of RIPK2. Cellular lysates were collected and cleared. Streptavidin beads were used to pull down RIPK2 via SBP tag within the NTAP tag. Samples were analyzed by Western blotting. C, UbcH7−/− HEK 293T cells were transiently transfected with HA-ubiquitin, NTAP-RIPK2, FLAG-ITCH, FLAG-ITCH C830A, and Myc-UbcH7. NTAP was precipitated under stringent washing conditions (1% SDS, 2 m NaCl). Samples were run on SDS-PAGE next to WT samples that were similarly transfected. Samples were subjected to Western blot analysis as indicated. Results shown are representative of three independent experiments. D, HA-tagged ubiquitin, NTAP-tagged RIPK2, and FLAG-ITCH were transiently transfected into UbcH7−/− HEK 293T cells with CRISPR-resistant UbcH7 WT, F63N, or F63S. RIPK2 was pulled down with streptavidin beads. Samples were washed and subjected to SDS-PAGE and immunoblotting as indicated.