File S1 - BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

Supporting Figures. Figure S1. Constitutively active form of BRK mutant (BRK-YF) shows maximum kinase activity. HEK 293 cells were transiently transfected with empty control vector (-) GFP-BRK-WT, GFP-BRK-KM or GFP-BRK-YF followed by immunoblotting analysis using anti-GFP and anti-phosphotyrosines antibodies. Figure S2. The knock down of BRK in SKBR3 cells restores DOK1 protein level. (A) SKBR3 cells were treated with EGF (100 ng/ml) for 0, 5, 10, 15 and 30 minutes and then subjected to immunoblot analysis for the detection of phosphotyrosines and β-tubulin (as a loading control). (B and C) SKBR3 and stable BRK knock down SKBR3 cells were treated with or without EGF (100 ng/ml) for 15 minutes. Total cellular proteins were determined from the cell lysates by performing immunoblot analysis with anti-BRK and anti-DOK1 antibodies. β-actin served as a loading controls and the DOK1 expression was quantified and shown in a bar diagram. Figure S3. Dok1 is not ubiquitinated in the absence of BRK. HEK293 cells were transiently co-transfected with GFP-Dok1, HA-ubiquitin and empty myc vector and incubated in the presence or absence of the proteasomal inhibitor, MG132 (10 µM) for 8 hours. Cell Lysates were subjected to immunoprecipitation with anti-Dok1 antibody and immunoblotting was performed with antibodies against HA and Dok1 (top panel). Total cell lysates were subjected to immunoblotting with antibodies against Dok1, BRK and β-tubulin as loading control. Figure S4. Dok1 inhibits BRK-induced cell proliferation in MDA-MB 231 cells. (A&B) MDA-MB 231 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. Figure S5. Dok1 inhibits BRK-induced cell migration in MDA-MB 231 cells. (A & B) MDA-MB 231 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell migration based on the healing of the wound area. The percentage of open area at 24 hours is plotted. (C & D) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: *P≥0.05 and **P≥0.001. (PPT)