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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: J Pathol. 2014 Jul 9;234(1):60–73. doi: 10.1002/path.4375

Figure 2.

Figure 2

Smoke-induced MUC1-C/p120ctn interaction depends on MUC1 glycosylation in polarized HBE cells. (A) HBE cells exposed to smoke (Smk) were harvested at indicated time points, IP’d using anti-MUC1-C and probed with VU4H5, anti-p120ctn and anti-MUC1-C. IgH bands served as the loading control. Densitometric quantitation of 400kDa MUC1-N and MUC1-C bound p120ctn in Smk-treated cells was normalized to untreated Ctrl (designated 0h as 1-fold) and reported as mean ± SEM fold changes. **p < 0.01, Smk-treated cells versus Ctrl. (B–C) HBE cells were stained by immunofluorescence after exposure to Smk and smoke-free medium (Ctrl) for 4h. (B) Immunostaining with anti-MUC1-C (red, top panels) and anti-p120ctn (green, middle panels) is shown. White boxes in middle panels indicate p120ctn at intercellular areas, which remains intact in Ctrl cells but predominantly lost in Smk-exposed cells. Merged MUC1-C and p120ctn images (generating yellow signals, bottom panels) demonstrate colocalization of MUC1-C and p120ctn in the cytoplasm and basolateral membranes (white arrowheads) after smoke exposure. (C) Staining with anti-MUC1-N (red, top panels) and anti-Tn (anti-GalNAc, green, middle panels). Glycosylated MUC1-N (MUC1-N-Tn, bottom panels) is indicated by overlaying of MUC1-N and Tn images generating yellow signals (yellow arrowheads). Cell nuclei were visualized with DAPI (blue). Scale bar represents 50μm. (D) HBE cells were preincubated with TB [20ug/ml tunicamycin (N-glycosylation inhibitor) and 2mM benzyl-α-GalNAc (O-glycosylation inhibitor)] or vehicle control (DMSO) overnight and exposed to Smk and Ctrl medium for 4h in the presence of TB. MUC1-C IP was analyzed with MUC1-N, MUC1-C and p120ctn antibodies. After TB treatment, anti-MUC1-N recognized the 150kDa and 230kDa isoforms of MUC1-N, while anti-MUC1-C recognized MUC1-C bands between 20kDa and 22kDa. Equal loading was confirmed with IgH. Densitometric quantitation of p120ctn IP’d by MUC1-C in Smk and/or TB-treated cells was normalized to untreated Ctrl (designated as 1-fold) and reported as mean ± SEM fold change. **p < 0.01, Smk and/or TB-treated cells versus untreated Ctrl.