LYN binding to the PRR of CBL is enhanced in cells expressing CBL mutants. (A) Schematic of CBL variants with mutation of the RING domain (C384Y) in combination with the TKB domain (G306E), PRR (ΔPRR), or C-terminal phosphotyrosine sites (Y700F/Y731F/Y774F). RING domain and phosphotyrosine site mutations are indicated by asterisks. (B) Competition between CBL WT (dTomato) and CBL double mutants (GFP) expressed in 32D-Cbl^KO cells. Mean and standard deviation (SD) of triplicate values are depicted. Statistical significance was determined by 2-tailed Student t test comparing CBL C384Y to CBL WT and each CBL double mutant (*P < .05; **P < .001). (C) MS analysis of V5 IP samples from 32D-Cbl^KO cells expressing V5-tagged CBL WT, Y371H, C384Y, or R420Q. Volcano plot showing log₁₀P values and log₂ fold change in protein abundance between CBL mutants (pooled data from Y371H, C384Y, and R420Q) and CBL WT samples. (D) WB for V5, LYN, and vinculin (VCL) in anti-V5 IP samples and whole-cell lysates (WCL) from 32D-Cbl^KO cells expressing V5-tagged CBL WT, Y371H, C384Y, and R420Q. (E) WB for V5, LYN, and vinculin (VCL) in anti-V5 IP samples and whole-cell lysates from 32D-Cbl^KO cells expressing V5-tagged CBL WT, C384Y, C384Y/G306E, C384Y/ΔPRR, or C384Y/3YF. L, linker; pY, phosphotyrosine; UBA, ubiquitin-associated.