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. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: Eur J Immunol. 2019 Sep 30;50(1):48–55. doi: 10.1002/eji.201948204

Fig 2. TAM receptor Tyro3 phosphorylates tyrosine residues 133 and 363 of Cbl-b.

Fig 2.

(A) HA-tagged Tyro3 was incubated with truncated Cbl-b (TKB+RF), E1, E2, ubiquitin, and ATP in vitro for 1 hour and immunoblotted with anti-HA antibody. (B) Truncated Cbl-b (TKB+RF) was incubated with or without recombinant Tyro3, Axl, or Mer and with or without ATP in vitro and immunoblotted with anti-phosphotyrosine antibody. (C) LAKs were IL-2-starved for 18 hours and serum-starved for 4 hours followed by stimulation with Gas6 for various time points or (D) followed by stimulation with or without Gas6, BMS 777607 with Gas6, or anti-NK1.1 (PK136) with or without Gas6 for 60 minutes. Lysates were de-ubiquitinated with DUB USp2core, followed by immunoprecipitation with anti-phosphotyrosine and immunoblotting with anti-Cbl-b antibodies. (E) 10XHis-tagged Y→F Cbl-b point mutants were incubated with Tyro3 and ATP, followed by immunoblotting with anti-phosphotyrosine antibody. Y3F refers to Y106/133/363F triple mutant. (F) 10XHis-tagged Y→F Cbl-b point mutants were incubated with Tyro3, E1, E2, ubiquitin, and ATP for 1 hour and immunoblotted with anti-ubiquitin antibody. One representative of three independent experiments is shown.