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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Biochim Biophys Acta. 2010 Jun 30;1803(10):1198–1205. doi: 10.1016/j.bbamcr.2010.06.007

Figure 2.

Figure 2

I. Direct phosphorylation of galectin-3 by c-Abl in vitro. Approximately 1 μg of recombinant wild-type or mutated galectin-3 proteins used as substract for active c-Abl in in vitro assay. 100 μl kinase assay buffer containing 100 μM ATP and 25 μCi of [g-32P] ATP with recombinant galectin-3 and active c-Abl was incubated for 20 min at 30°C. After reaction was stopped with sample buffer, boiled samples were subjected to phosphopeptide map analysis as described in Materials and Methods.

II. Phosphorylation of galectin-3 by c-Abl in vivo. Wild type galectin-3 protein was expressed in SK-Br-3 cell with constitutively active c-Abl PP (lane 1,2) and kinase dead c-Abl KM (lane3). Cells in lane 2 were treated with 10 μM of c-Abl inhibitor STI571. Galectin-3 was immunoprecipitated using anti-galectin-3 (TIB166) antibody and loaded on gel. The samples were resolved using 10% SDS-PAGE and immunoblotted with anti-Gal-3 (HL31) (top) and anti-phosphotyrosine antibodies (bottom).

III. Dose-dependent inhibition of c-Abl activity by STI571. SK-Br-3 cells were co-transfected with c-Abl PP and wild type (lane 1-6) or Y107F (lane 7-12) galectin-3. STI571 was added to cell medium overnight. Cells were lysed and galectin-3 was immunoprecipitated using anti-Gal-3 (TIB166) antibody and loaded on gel. The samples were resolved using 10% SDS-PAGE gel and immunoblotted with anti-Gal-3 (HL31) (top) and anti-phosphotyrosine antibody (middle). To confirm equal loading of immunoprecipitated proteins 15% of IP mixture was run on 10% SDS-PAGE and visualized with Coomassie blue stain. The normalized integrated intensity was calculated as Band integrated intensity/Lane normalization factor, anti-galectin-3 blot was used as reference channel. The band having the highest integrated intensity is assigned a normalization factor of 1.0. This reference band is then used to calculate the normalization factor for all other selected lanes by dividing the band integrated intensity by the integrated intensity of the reference band. Then the normalization factor was used to calculate the normalized integrated intensity.