Fig 1: CBLock inhibits RTK-mediated GOF properties in CBL mutants(A) Proliferation of CBL mutant-expressing cells treated with vehicle (water) or 5 μM cp-CBLock peptide were analyzed after 72 h using Cell Titer-Glo and represented as percentage of cells treated with vehicle. The data points are presented from three independent experiments while the error bars represent ±SD (n = 3 independent experiments). (B) Percentage of cells in different phases of cell cycle are represented. MOLM-14 cells were treated with 5 μM cp-CBLock-WT or cp-CBLock-I10A for 72 h, followed by PI staining and cell-cycle profile analysis by FACS. The data are presented as mean of three independent experiments while the error bars represent ±SD (n = 3 independent experiments). (C) Expression of c-Caspase 3, c-PARP, and Actin (loading control) from lysates of K562 and MOLM-14 cells treated with different concentrations of cp-CBLock. (D) Foci-formation assay using NIH3T3cells stably expressing CBL-WT or CBL-Y371S in the presence of either GFP-CBLock-WT or GFP-CBLock-I10A. (E) Quantification of (D), data are presented from three independent experiments while error bars represent ±SD (n = 3 independent experiments). (F) Co-immunoprecipitation assay of HEK293 cell lysates overexpressing Myc-tagged CBL-Y371S or the EV (−) along with GFP, GFP-CBLock-WT, or GFP-CBLock-I10A as indicated in the figure. Anti-EGFR immunoprecipitants and cell lysates were analyzed by immunoblotting with anti-Myc–tag, anti-EGFR, anti-GFP, and anti-Actin antibodies as indicated. (G) Co-immunoprecipitation assay of HEK293 cell lysates overexpressing Myc-tagged CBL variants or the EV (−) along with either GFP-CBLock-WT or GFP-CBLock-I10A. Anti-Myc–tag antibody immunoprecipitants and cell lysates were analyzed by immunoblotting with CBL-pY731, anti-Myc–tag, anti-GFP, and anti-Actin antibodies as indicated. (H) Heatmap of mass spectrometry data comparing CBL interactomes in MOLM-14 cells in the presence of vehicle (water) or 5 μM cp-CBLock. MOLM-14 cells were treated with vehicle or cp-CBLock for 48 h and cell lysates were immunoprecipitated using anti-CBL antibody- or mouse IgG-conjugated agarose beads followed by mass spectrometric analysis. Red and blue represent strong- or no/weak-interaction, respectively. The experiment was performed in three biological replicates. (I) Proliferation of MOLM-14 cells transfected with control siRNA (cont si), MRG-1 (CITED2) siRNA (MRG-1 si), DYNLL1 siRNA (DYNLL1 si), BUD31 siRNA (BUD31 si), RPL34 siRNA (RPL34 si), or Ikaros siRNA (Ikaros si) and analyzed after 48 h using Cell Titer-Glo and represented as percentage of cells knocked down with cont si. Data are presented from three independent experiments while error bars represent ±SD (n = 3 independent experiments). The Actin panel is a sample control in (C), (F), and (G).