Western blot analysis of muscarinic 3 receptor. Extraorbital lacrimal glands (LGs; n = 4) were lysed and processed by using different antibodies/serum at all ages. Representative images are shown; the number after "LG" indicates the ID of LG used for that particular Western blot. (A) Representative Western blot of LG2 lysates blotted with M3R antibody. (B through D) Representative Western blot of LG lysates blotted with C57BL/6 (B, LG3), γDKO (C, LG4), or CD25KO (D, LG3) sera used to generate the bar graph in E of net band densities (arbitrary values). 8W, 8 weeks; 16W, 16 weeks. (E) Mean ± standard deviation M3R net band densities normalized by β-actin band densities in Western blots of LG lysates blotted C57BL/6, γDKO, or CD25KO sera at different ages. (F) Co-immunoprecipitation studies. LGs were co-immunoprecipitated with either 4 or 8 μg of muscarinic 3 receptor antibody (lanes 2 and 3) and blotted with CD25KO-16W serum. Excess proteins that were not precipitated (Wash 1 (W1) and wash 2 (W2), lanes 4 and 5, respectively) were included in the gel and blotted as negative controls. LGs and brain lysates (lane 1 and 6) that were not immunoprecipitated were included in the gel as positive controls.