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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: J Cell Physiol. 2010 Sep;224(3):649–657. doi: 10.1002/jcp.22163

FIGURE 1.

FIGURE 1

FIGURE 1

FIGURE 1

Muc4 modulates ErbB2 signaling potential . A. Muc4 forms a stable complex with phosphorylated ErbB2. Proteins from RIPA extracts of A375 Muc4-transfected cells with or without Muc4 (48 h) were subjected to immunoprecipitation with anti-rCpep, a Muc4 pAb targeting the cytoplasmic portion of Muc4, or with the pre-immune serum (−). Immunoprecipitates were immunoblotted with anti-Muc4, anti pY1248-ErbB2, and anti-ErbB3 antibodies. The control immunoprecipitation lane (+) in the ErbB3 immunoblot was carried out with the anti-ErbB3 (C-17) polyclonal antibody. The input control is an immunoblot of the lysates with anti-Muc4 and anti β-actin antibodies. B. Muc4 expression augments ErbB2 phosphorylation signal magnitude significantly (p=0.0002) without changing ErbB2 receptor levels. A375 Muc4-transfected (Rep3 clone) cells with or without Muc4 (48 h) and starved (0.1% FBS) for 24 h were immunoblotted in triplicates with anti-Muc4, anti ErbB2-Y1248, anti-ErbB2, and anti β-actin antibodies. Quantitative analysis of the ErbB2-Y1248 signal intensity is also shown. The signal intensity was calculated using the ImageJ NIH densitometry software. The ErbB2-Y1248 signal was measured, normalized with the β-actin signal, and expressed as the mean ± standard deviation for a series of at least 3 experiments. Student’s t tests were used to compare mean values as appropriate. P values < 0.05 were considered to represent significant differences. C. Muc4 expression stabilizes ErbB2 phosphorylation signal under heregulin ligand (HRG-1β) treatment. A375 Muc4-transfected (Rep 3 clone) cells with or without Muc4 (48 h) and starved (0.1% FBS) for 24 h, were incubated at 37°C with HRG-1β [50 nM] for the indicated times. Quantitative analysis of ErbB2-Y1248 immunoblot band density is shown. Using densitometry software (ImageJ NIH), the signal was measured, normalized with β-actin signal, and then expressed as the mean ± standard deviation for a series of at least three experiments. Circles represent Muc4-Off cells treated with ligand, and squares, Muc4-On cells treated with ligand.