Figure 4
Subcellular Fractionation of α1, αv, β1, and β5 integrins in neuroblastoma cells highly expressing TRPM2. (A) Subcellular separation of two clones of SH-SY5Y KO cells reconstituted with TRPM2 (KO1-M2, KO2-M2) into cytoplasmic, membrane, and nuclear fractions was performed as described in "Materials and Methods". Western blotting was performed with fractionated samples loading equivalent amounts per lane (10 ug/lane). Blots were probed with antibodies to α1 (ITGA1), αv (ITGAV), β1 (ITGB1), β5 (ITGB5) integrins. Blots were also probed with antibodies to GAPDH (cytoplasmic marker), Na,K-ATPase α (plasma membrane marker which regulates Na and K ions and cell volume) and Lamin A/C (nuclear marker) as controls. Four experiments were performed with similar results and the results of one are shown. α1, αv, and β1 were predominantly found in the membrane fraction, and β5 in the cytoplasmic. Full length gels for these Western blots are shown in Supplementary Fig. S4. Membrane fractionations of Scr1-V, Scr2-V, KO1-V, KO2-V, KO1-M2, KO2-M2, KO1-E960D, KO2-E960D cells are also shown on Supplementary Fig. S4. (B) Flow Cytometry of α1, αv, β1, and β5 integrin expression on the surface of non-permabilized SH-SY5Y KO cells (KO1-V, KO2-V) and KO cells expressing TRPM2 (KO1-M2, KO2-M2). Fluorescent intensities (X-axis) of integrins α1, αv, and β1 were increased on the surface of KO-M2 compared to KO-V cells. This experiment was performed twice with similar results and results of one experiment are shown.
