Figure 5.

Expression of markers specific for cholinergic neurons. M17 cells were grown without or with 10 μM RA for 72 hours to induce differentiation. The cells were washed and solubilized in sample buffer and analyzed by Western blotting for (A) choline acetyltransferase (ChAT), (B) M1 muscarinic acetylcholine receptor (mAcR), and (C) nicotinic acetylcholine receptor α 7 (nAcR a-7). Either β-actin or GAPDH was used as a house keeping protein marker to show equal protein loading of gels. The relative amount of each marker protein was quantified by densitometric analysis using Image J program (NIH public domain program, http://rsbweb.nih.gov/ij/index.html). Differences in marker proteins in differentiated vs. undifferentiated cells were assessed in terms of % normalized optical density in differentiated cells compared to undifferentiated (no RA) controls as shown in the bottom panel (D). Normalized optical density of undifferentiated control was 100% for each type of marker protein. ND = not detected and as such could not be quantified. For M1 mAcR, the difference between undifferentiated vs differentiated was not statistically significant (student t test). n=4.