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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 3.

Figure 3

Smoke-induced MUC1-C glycosylation modulates MUC1-C tyrosine phosphorylation (TyrP) in polarized HBE cells. (A–B) HBE cells were preincubated with TB or vehicle control (DMSO) overnight before treating with Smk or Ctrl medium for 4h in the presence of TB. (A) Cell lysates were analyzed by WB probed with TyrP (PY100 or PY20) antibodies. Blots were stripped and reprobed with MUC1-C antibody. Bands recognized by PY100 and PY20 overlapped with each other and with MUC1-C. Equal loading was confirmed with GAPDH. Densitometric quantitation of MUC1-C-TyrP (PY100) and MUC1-C-TyrP (PY20) after Smk and/or TB treatment was normalized to untreated Ctrl (designed as 1-fold) and reported as mean ± SEM. **p < 0.01, Smk and/or TB-treated cells versus untreated Ctrl. (B) Immunostaining with anti-MUC1-C (red, top panels), anti-PY100 (green, middle panels of left two lanes) or anti-PY20 (green, middle panels of right two lanes). Merged MUC1-C and PY100/PY20 images (generating yellow signals, bottom panels) demonstrate smoke-provoked MUC1-C-TyrP (yellow arrowheads). Cell nuclei were visualized with DAPI (blue). Scale bar represents 50μm.