Figure 3

Plectin is the putative target of PCS2. (A) Outline of the benzophenone-PCS2-coated magnetic bead pull-down approach used for proteomics based target identification (see Supplementary Fig. S5 for peptoid characterization). (B) Target pull-down from H358 cell surface shows unique protein bands at ~500 kDa, which was not found in the leftover cell lysate and control pull-down fraction. The standard proteomics analysis indicated that plectin protein was the potential hit in this region. The gel is cropped, and full length blots are presented in Supplemental Fig. S8. Note: This assay was repeated 3 times, and in all cases plectin protein was pulled down as most likely target (See Supplementary Proteomics Dataset S1, 2, 3 and 5), while MYH9, or AHNAK proteins also could have been possible targets (See Supplementary Proteomics Dataset S3 and 4). (C) Western blot of the PCS2-target pulldown in HBEC3KT, H1155, and H358 cells, immunostained with Plectin, MYH9, and AHNAK antibodies. Only plectin was detected in the PCS2-bound fraction of H358 while no PCS2 binding occurred in HBEC3KT or H1155, indicating the target of PCS2 is plectin. The anti-plectin antibody incubated during H358 cell binding to benzophenone-PCS2-coated magnetic beads reduced the intensity of the plectin band, further confirming plectin as the target. In addition, benzophenone-PCS2-coated magnetic beads were incubated with H358 lysate to see whether cytosolic proteins would nonspecifically attach to the bead surface, but no bands were observed, indicating this has not occurred. MYH9 and AHNAK antibodies were used as non-specific controls, as they were identified, non-reproducibly, as potential targets in a single pull-down replicate (Supplementary Proteomic Dataset S3, S4). But none of the bound fractions display significant bands. The blots are cropped, combined from multiple gels (that were repeated at least 3 times), and full length blots are presented in Supplemental Fig. S9. (D) PCS2-coated magnetic-beads bound fractions of H358, H1693, H460, and H1975 cells have higher expression of plectin (PLEC) compared to unbound and unsorted fractions. (E) The inclusion of anti-plectin antibody significantly decreases the percentage of cells binding to PCS2-coated magnetic-beads on those H358, H1693, and H460 cells. Error bars represent standard deviation between triplicates. *Represents p value < 0.05.