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Files in this Data Supplement:
Fig. S1. Subcellular fractionation of cells expressing the ALK-FH-derived proteins. Hypotonic lysates of cells expressing the indicated ALK-FH-derived proteins or non-transfected cells (Mock) were separated into cytosolic (C) and membrane pellet (M) fractions by ultracentrifugation with total lysate (T) as control. Fractions were subjected to western blot analysis using the anti-HA tag antibody (3F10). Blots were then re-probed with anti-transferrin receptor (TfR, membrane marker) and anti-stathmin (cytosolic marker) antibodies.
Fig. S2. Basal and induced ERK phosphorylations triggered by activation of the myrIA-FH and cytoIA-FH proteins were a result of their intrinsic kinase activities. (A) HEK 293 cells stably transfected with the indicated ALK-FH-derived constructs were treated or not with the dimerizer during 30 minutes. Cells were lysed in RIPA buffer and 100 mg of proteins were then immunoprecipitated using the 1.5 mg of mouse anti-HA tag antibody (12CA5). Western blot analysis was performed using either the rat anti-HA tag antibody (3F10) or the phosphotyrosine (4G10) antibody. (B) PC12 cells were submitted to the same protocol as described in Fig. 4C. As control, non-transfected cells (Mock) or cells expressing the myrIA-FH or cytoIA-FH kinase-defective mutants were treated or not with the dimerizer during 30 minutes. Cells were lysed and submitted to western blot analysis as described in Fig. 2C,D.
Fig. S3. U0126 totally inhibits basal and induced ERK 1/2 phosphorylations in cells expressing the tmbIA-FH and myrIA-FH proteins. PC12 cells were submitted to the same protocol as described in Fig. 5. Cells were treated or not with the dimerizer for 30 minutes. Cells were lysed and submitted to western blot analysis as described in Fig. 2C,D.
Fig. S4. Wortmannin inhibits basal and induced AKT phosphorylations in cells expressing the cytoIA-FH protein. PC12 cells were treated as described in Fig. 7. Cells were treated or not with the dimerizer for 30 minutes. Cells were lysed and submitted to western blot analysis. The P-AKT densitometry quantification was performed using the Odyssey Imaging System on three independent experiments. ***, P<0.005 (Student’s t-test).
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