Fig. 7

Recombinant Chk-GFP inhibits Src and forms complex with it in DLD1 cells. DLD1 cells were transduced with lentiviral vectors that directed expression of GFP or Chk-GFP under the control of 5 mg/ml doxycycline (dox). The transduced cells, referred to as DLD1-GFP or DLD1-Chk-GFP were examined for Src activity and formation of protein complexes containing Chk-GFP and Src. a Western Blot of cell lysates from the transduced cells in the presence and absence of Dox. Lysates (15–20 μg) were probed with anti-Chk and anti-GFP antibodies. Tubulin was used as the loading control (anti-tubulin) and the recombinant 52 kDa isoform of Chk was loaded as the positive control. b Immunofluoresence analysis of the transduced DLD1 cells before and after induced expression of GFP or Chk-GFP. The nuclei were stained with DAPI. c Src was immunoprecipitated from the transduced cell lines in the presence or absence of Dox, using anti-Src antibody. Its specific enzymatic activity in the immunoprecipitates was determined using Src-optimal peptide as the substrate. d Western blot of immunoprecipitated Src which was used to monitor its enzymatic activity. Anti-pY527, anti-pY416 and anti-Src antibodies were used to detect phosphorylation and presence of Src. Recombinant Src (truncated) was used as a positive control. e Co-immunoprecipitation of Src and Chk-GFP. GFP trap magnetic beads (Chromotek) were used to immunoprecipitate Chk-GFP from the transduced +/− dox cell lysates. Western blotting with anti-Src antibody was conducted to detect Src and Chk-GFP in the immunoprecipitated, the unbound fraction and the wash fractions. L = Original lysate, UF = Unbound fraction, W1 = Wash 1, W 3 = Wash 3, B = Proteins bound to the beads eluted with SDS sample buffer. f Soft agar colony-formation assay of GFP-expressing and Chk-GFP-expressing DLD1 cells. Expression of GFP and Chk-GFP was induced by doxycycline for 48 h prior to the assay. Experiment was performed in triplicates (n = 3 wells). Colonies were stained by 0.005% crystal violet for ~1 h. Images were taken using Biorad’s image lab software. Left panels colonies formed on soft agar by the transduced DLD1 cells. Right panels statistical analysis of colony-formation result. ImageJ software was used to count and define the sizes of colonies (Large: ≥ 20 pixels; Medium: 11–20 pixels; Small: 1–10 pixels). The colony number of each sample was demonstrated in the left panel, while the colony size was demonstrated in the right panel. The values represent the mean ± S.D. (n = 3 wells). *p > 0.05, **p < 0.05