Figure 4
Plectin is found on H358 cell surface and plectin antibody binding identified a subpopulation of H358 cells with CSC-like characteristics in a similar manner to PCS2. (A) Plectin antibody-coated magnetic-beads bind to a similar percentage of cells when compared to PCS2-coated magnetic-bead binding in 4 lung cancer lines (H358, H1693, H460, H1975) and had no detectable binding on PCS2-non-binding lung cancer line H1155 and normal HBEC3KT. In comparison, GST antibody-coated and non-binding control compound-coated beads do not bind to any of the cell lines tested. (B) The Pierce Cell Surface Protein Isolation Kit (Thermo Fisher) biotinylated only outer cell surface proteins, isolated and separated those from cytosolic proteins. The plectin protein is significantly detected in the cell surface fraction of H358 cells, and not significantly in HBEC3KT, H1155, or H358 without the initial cell surface biotinylation component, while plectin is detected in all 3 lines in the cytosolic fraction. β-actin is used as a cytosolic-specific marker for comparison, which did not appear on the cell surface fraction indicating the purity of the cell surface fraction. The blots are cropped, combined from multiple gels (that were repeated at least 3 times), and full length blots are presented in Supplemental Fig. S10. (C) Plectin antibody-coated magnetic-bead bound fraction from H358 have higher expression of PLEC, ALDH1A3 and SOX2 compared to the unbound fraction. Error bars represent standard deviation between duplicates. *Represents p value < 0.05.
