immunoprecipitation pulldown hek 293t  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

doi: 10.1074/jbc.RA117.000408

Figure Lengend Snippet: Phosphorylation disrupts UbcH7 binding. A, the structure of the E6AP–UbcH7 complex (1C4Z) is shown to the left of the panel series with the E6AP HECT domain colored cyan and UbcH7 colored yellow. The upper panel highlights the E6AP–UbcH7 interface near E6AP residue Ser-638 at the H7 helix, which forms a side chain to main chain hydrogen bond with UbcH7 residue Phe-63 at the L1 loop. To model the ITCH–UbcH7 interface, the structure of the ITCH HECT domain (3TUG, colored green) is superimposed onto the E6AP HECT domain within the E6AP–UbcH7 complex and shown at the upper right panel. Similar to the E6AP residue Ser-638, the ITCH residue Ser-687 is predicted to form the same side chain to main chain hydrogen bond with the UbcH7 residue Phe-63. Mutation of Ser-687 to Asp (lower left) or phosphorylation of Ser-687 (lower right) would lead to loss of the hydrogen bond and significant steric hindrance at the E2–E3 interface, thus severely impairing ITCH–UbcH7 interaction. B, HEK 293T cells were transfected with FLAG-ITCH and FLAG-ITCH S687D alone or with Omni-UbcH7 for co-precipitation. FLAG-ITCH was immunoprecipitated (IP) in each sample. After stringent washing of IP samples, lysate and IP samples were ran on SDS-PAGE, transferred to nitrocellulose, and probed as indicated. Similar results were obtained in three independent experiments. C, HEK 293T cells were stimulated with 1 μg/ml TNFα for the indicated time points and UbcH7 was immunoprecipitated. After gentle washing, lysate and IP samples were processed by Western blotting. Membranes were probed as indicated. Similar results were obtained in two independent experiments. D, Biacore SPR sensorgrams for binding of UbcH7 to wildtype or S687D mutant ITCH.

Article Snippet: Cell culture, transient transfection, and immunoprecipitation/pulldown HEK 293T (CRL-1573; American Type Culture Collection) cells were grown in DMEM 10% SCS with 1% penicillin/streptomycin.

Techniques: Phospho-proteomics, Binding Assay, Residue, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Gentle, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

doi: 10.1074/jbc.RA117.000408

Figure Lengend Snippet: 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.

Article Snippet: Cell culture, transient transfection, and immunoprecipitation/pulldown HEK 293T (CRL-1573; American Type Culture Collection) cells were grown in DMEM 10% SCS with 1% penicillin/streptomycin.

Techniques: Phospho-proteomics, Transduction, CRISPR, Negative Control, Clone Assay, SDS Page, Transfection, Ubiquitin Proteomics, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

doi: 10.1074/jbc.RA117.000408

Figure Lengend Snippet: 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.

Article Snippet: Cell culture, transient transfection, and immunoprecipitation/pulldown HEK 293T (CRL-1573; American Type Culture Collection) cells were grown in DMEM 10% SCS with 1% penicillin/streptomycin.

Techniques: Phospho-proteomics, Transfection, Ubiquitin Proteomics, Western Blot, Negative Control, SDS Page