Author Manuscript NIH PA Author Manuscript NIH PA Bosutinib SRC inhibitor Author Manuscript streptomycin sulfate. NR 6m cells, a subclone of NR 6 that stably expresses the EGFRvIII, were provided by Dr Darrel Bigner and were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin sulfate, and 750 g/ml G 418. CHO cells were transfected with various constructs using FuGENE 6, whereas HEK 293T cells were transfected using calcium phosphate. Following transfection, cells were grown to 70% confluence and starved overnight in DMEM supplemented with 0.5% FBS. Then, cells were treated as described in the figure legends before the preparation of cell lysates. NIH 3T3 cells were transfected with the EGFRvIII, Y1045F EGFRvIII, HA Cbl b, C373A HA Cbl b, or empty vector controls as indicated using Effectine.
A day after the transfection, the cells were split 1:3 and grown for 14 Celecoxib Celebra days in selection medium containing either 600 g/ml Zeocin alone or a combination of 600 g/ml Zeocin and 600 g/ml G 418. Stable clones were pooled and foci assays were performed at passage 3 by plating 1×106 cells per 100 mm tissue culture dish. Cells were incubated 1 2 weeks, fixed with 10% methanol, 10% acetic acid solution for 15 min, and stained with 20% ethanol, 0.4% crystal violet for 5 min. Immunoblotting and immunoprecipitation To harvest proteins, cells were washed twice in ice cold DPBS containing 200 M sodium orthovanadate and then lysed in ice cold lysis buffer, 2 mM sodium orthovanadate, and protease inhibitors. The lysates were cleared of debris by centrifugation at 16 000 g for 10 min at 4.
Supernatant protein concentrations were determined using a BioRad protein assay. For immunoblotting, lysates were boiled in loading buffer for 5 min. For immunoprecipitation, lysates containing 500 g protein were incubated with either a mouse monoclonal anti EGFR antibody and Protein A/G agarose beads or HA affinity matrix overnight at 4 with tumbling. Immune complexes were washed five times in cold lysis buffer, resuspended in 2× loading buffer and boiled for 5 min. The proteins were resolved by SDS PAGE and transferred to PVDF membranes. Membranes were probed with either rabbit polyclonal anti EGFR, rabbit polyclonal anti phosphotyrosine 1045 EGFR, rabbit polyclonal anti Cbl, rabbit polyclonal anti Cblb, goat polyclonal anti Cbl c, mouse monoclonal anti HA, mouse monoclonal anti GFP, mouse monoclonal anti Tubulin, or peroxidase linked anti phosphotyrosine antibodies.
Horse radish peroxidase linked donkey anti rabbit, donkey anti mouse, or rabbit anti goat immunoglobulin was used with SuperSignal to visualize the blots. Immunoblots were quantified on a PC computer using the public domain NIH Image program. Davies et al. Page 10 Oncogene. Author manuscript, available in PMC 2008 March 25. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Immunofluorescence and confocal microscopy NR 6m cells or pooled clones of NIH 3T3 cells expressing the EGFRvIII or Y1045F EGFRvIII were plated at 2×104 cells/ well in four well chambered coverslips and incubated overnight. Then, the NR 6m cells were incubated for 3 h with 100 g/ml cycloheximide and either 30 M AG 1478 or 0.1% DMSO. Following a rinse with PBS, both NR 6m and NIH 3T3 cells were fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. The chambers were rinsed three times with PBS,