Purification of GST fusion proteins and GST pull-down assays from extracts of HeLa cells or MCF7 transiently expressing GFP fusion proteins were performed as described [3 (link), 20 (link)] using 15-30 μg of GST or GST-EB1 beads per experimental condition. Immunoblotting was using rabbit anti-MTUS1 (ARP44419, Aviva Systems) (1:1000) or monoclonal anti-GFP antibodies (clone 7.1/13.1, Roche) (1:3000).
For in vitro interaction, chemically synthesized peptides CN45 and CC coupled to FITC were purchased from GL Biochem (Shanghai, China). Purified peptides were incubated for 1h at room temperature with GST- or GST-EB1 beads in 50mM HEPES containing 150mM NaCl, 0.01% Triton X100 (pH 7.4) then washed in the same buffer. Interaction was assessed by FITC fluorescence measurement using Fusion Universal Microplate Analyzer (Packard BioScience) and with Typhoon™ system (Amersham Biosciences) following 15% SDS-PAGE.
For immunoprecipitation, cell lysates were incubated for 2h at 4°C with 4μg of mouse monoclonal anti-GFP (Roche), or mouse monoclonal anti-mCh (Clontech) antibodies prior to incubation with G protein-sepharose beads. Bound proteins were detected by immunoblotting as described above.
For in vitro interaction, chemically synthesized peptides CN45 and CC coupled to FITC were purchased from GL Biochem (Shanghai, China). Purified peptides were incubated for 1h at room temperature with GST- or GST-EB1 beads in 50mM HEPES containing 150mM NaCl, 0.01% Triton X100 (pH 7.4) then washed in the same buffer. Interaction was assessed by FITC fluorescence measurement using Fusion Universal Microplate Analyzer (Packard BioScience) and with Typhoon™ system (Amersham Biosciences) following 15% SDS-PAGE.
For immunoprecipitation, cell lysates were incubated for 2h at 4°C with 4μg of mouse monoclonal anti-GFP (Roche), or mouse monoclonal anti-mCh (Clontech) antibodies prior to incubation with G protein-sepharose beads. Bound proteins were detected by immunoblotting as described above.