Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Gp135 possesses three N-glycosylation sites at the extracellular domain
  MDCK cells were mock-treated or transfected with Myc-tagged Gp135, or constructs expressing site-directed point mutation at the potential N-linked glycosylation sites (the 123th, 199th, 373th, and 383th asparagine (N) residues were mutated to glutamine (Q) individually). Lysates were immunoprecipitated by anti-Myc (9E10) mAb, and processed for Western blotting analysis by rabbit anti-Myc pAb. Please note that the N123Q mutant displays four intermediate N-glycosylated products. This pattern is in stark contrast to the other three mutants, which have only three intermediate N-glycosylated products, one less than the wild type protein. Furthermore, the mature form proteins of N199Q, N373Q, and N383Q all migrated faster than the wild type protein and the N123Q mutant. The positions of molecular weight markers, in kilodaltons, are indicated on the left.
• Supplemental Figure 2 - Predicted O-glycosylation regions in Myc-tagged Gp135 by NetOGlyc server 3.1
  The O-glycosylation threshold is arbitrarily set at 0.5 probability. A diagram of the Myc-tagged Gp135 expressing construct is depicted below. M, Myc-tag; TM, transmembrane domain; pink color shading region, a predicted membrane-distal O-glycosylation region; red color shading region, a predicted membrane-proximal O-glycosylation rich region; G, membrane proximal globular domain.
• Supplemental Figure 3 - Gp135 C-terminal Deletion of PDZ-binding motif disrupts EBP 50 binding
  (A) Bacterial lysates expressing the entire cytoplamic domain of Gp135 and a mutant devoid of the terminal PDZ binding motif fused to GST (GST-C and GST-dC respectively) were purified by glutathione beads. The purified GST fusion proteins were incubated with MDCK cell lysates, the beads collected by centrifugation and extensively washed. The pulled down materials were separated by SDS-PAGE and immunoblotted (IB) with rabbit anti-EBP50 and GST antibodies. (B) Cell lysates from MDCK stable clones expressing Myc-tagged full length (FL), m135dC (dC), or m135sC (sC) were processed for co-immunoprecipitation assay. The lysates were immunoprecipitated (IP) with Myc antibody (9E10) beads and immunoblotted (IB) with rabbit anti-Myc (A14) or anti-EBP50 antibodies. TL: total lysates.
• Supplemental Figure 4 - m135dC mutant protein is seen as punctates in the cytoplasm.
  Representative confocal section images of Myc-tagged full length Gp135 (FL), and m135dC (dC) expressing stable MDCK cells are shown. The focal section was selected at the apex of the nuclei; therefore, tight junctions are also visible in some cells. Green channel: anti-Myc staining; red:staining for tight junction marker ZO-1 and propidium iodide staining for nuclei. Bar: 10 μm