Fig 2: Hic-5 interacts with the RF of Cbl-c. A. Schematic diagram of Cbl-c constructs used to map the interaction with Hic-5. All constructs were HA-epitope tagged on the N-terminus. B. HEK293T cells were transfected with various HA- Cbl-c constructs and Hic-5 as indicated above panels. Immunoprecipitates (IP) or cell lysates (lysates) were immunoblotted (IB) as indicated to the right of the panels. All transfections were balanced with empty vector controls. GFP was transfected as a control for transfection efficiency and shown as a control for loading. MWs in kDa are shown to the left of the panels.

Fig 3: Mutation of the first zinc coordinating complex of the RF of Cbl-c disrupts Hic-5 binding. A. Sequence alignment of the RFs of Cbl, Cbl-b and Cbl-c. Amino acids in red show the nine amino acids that are divergent between the RF of Cbl-c and the RFs of Cbl and Cbl-b. The two positions highlighted in blue mark the amino acids shared between Cbl-c and Cbl-b that differ from Cbl. The two cysteines mutated to alanine in Figure 3D and E are indicated below the sequence. B. Schematic diagram of Cbl-c, Cbl-b and chimeric constructs with the swapped RF domains. Shaded boxes represent Cbl-c sequence. White boxes represent Cbl-b sequence. All constructs were HA-epitope tagged on the N-terminus. C. HEK293T cells were transfected with HA-Cbl-c, Cbl-b, or the chimeric proteins with swapped RF domains and Hic-5 as indicated above the panels. The Cbl proteins were immunoprecipiated with anti-HA beads and the immunoprecipitates (IP) or cell lysates (lysates) were immunoblotted (IB) as indicated to the right of the panels. All transfections were balanced with empty vector controls; GFP was transfected as a control for transfection efficiency and is shown as a control for loading. D. HEK293T cells were transfected with HA-Cbl-c, RF mutants of HA-Cbl-c, HA-Cbl-c ?RF, and Hic-5 as indicated above the panels. The Cbl-c proteins were immunoprecipitated with anti-HA beads and the immunoprecipitates (IP) or cell lysates (lysates) were immunoblotted (IB) as indicated to the right of the panels. All transfections were balanced with empty vector controls; GFP was transfected as a control for transfection efficiency and is shown as a control for loading. E. Purified recombinant GST-tagged Cbl-c RF proteins (WT, C351A or C366A mutants) bound to GSH-sepharose beads were added to a bacterial lysate expressing recombinant His-tagged Hic-5 or empty His vector as indicated above the panel. Proteins were precipitated with GSH-sepharose beads and immunoblotted (IB) as indicated to the right of the panels. Expression of His-Hic-5 in the bacterial lysates is shown in the lower panel. MWs in kDa are shown to the left of the panels.

Fig 4: Hic-5 colocalizes with Cbl-c.HeLa cells were transfected with CFP-Hic-5 and/or YFP-Cbl-c, fixed, and imaged using a Zeiss LSM 510 META confocal microscope with a 63X /1.3 NA immersion-oil Plan-Neofluar objective as described in the methods section. Images for each protein are shown in grey scale and for merged images in color. Panels 1–3 demonstrate the diffuse localization of YFP-Cbl-c and CFP-Hic-5. Panels 4–6 demonstrate the punctate localization of CFP-Hic-5, but not YFP-Cbl-c. In the merged images (panels 3 and 6) YFP-Cbl-c is shown in green, CFP-Hic-5 is shown in red and the overlapping signal is shown in yellow. To demonstrate that the punctate distribution of CFP-Hic-5 represents localization to focal adhesions, cells were stained using an anti-vinculin antibody as a marker of focal adhesions. Panels 7–9 demonstrate the colocalization of CFP-Hic-5 and vinculin in the punctate structures, consistent with colocalization in focal adhesions. Panels 10–12 show no colocalization of YFP-Cbl-c with vinculin. In the merged images for the vinculin staining (panels 9 and 12) YFP-Cbl-c and CFP-Hic-5 are shown in green, vinculin is shown in red, and the overlapping signal is shown in yellow. The imaris software was used for colocalization analysis, and Pearson’s correlation coefficient (PCC) was calculated for each image analysis on 5–10 cells for each combination (see text for results).

Fig 5: Hic-5 interacts with Cbl-c. A. HEK293T cells were transfected with HA-Cbl-c alone, Hic-5, or the combination of HA-Cbl-c and Hic-5 as indicated above panels. B. HEK293T cells were transfected with HA-Cbl-c alone, Trip-6, or the combination of HA-Cbl-c and Trip-6 as indicated above panels. In A and B, HA-Cbl-c was immunoprecipitated from the cell lysates. Immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. All transfections were balanced with empty vector controls. Green fluorescent protein (GFP) was transfected as a control for transfection efficiency and is shown as a loading control. C. Endogenous Cbl-c was immunoprecipitated from whole cell lysates from MB231 (which expresses Hic-5 but no Cbl-c) and CFPAC-1 (which expresses both Hic-5 and Cbl-c) cell lines and immunoblotted for Cbl-c and Hic-5. Immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. The arrow indicates immunoprecipitated Cbl-c and the asterisk indicates the immunoglobulin heavy chain. D. HEK293T cells were transfected with HA-Cbl, HA-Cbl-b, HA-Cbl-c alone, Hic-5 alone, or each HA epitope tagged Cbl protein with Hic-5 as indicated above the panels. Cbl proteins were immunoprecipitated and immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. The arrow indicates immunoprecipitated Hic-5 and the asterisk indicates an artifact band that includes the immunoglobulin heavy chain. MWs in kDa are shown to the left of the panels.