Protein concentrations in lacrimal gland lysates and lacrimal fluid were determined according to the Bradford protein protocol on a microplate reader (SOFTmax software; E-max microplate reader; Molecular Devices, Sunnyvale, CA).
Samples for polyacrylamide gels (SDS-PAGE) were prepared so that the same amount of protein could be loaded into each lane on the gel. Polyacrylamide gels at 12% were run according to standard protocols.
Protein loads were 100 μg per lane for analyses of TGF-β1 and EGF and 50 μg per lane for analyses of prolactin. Proteins were transferred onto nitrocellulose membranes and exposed to primary antibody overnight at 4°C (1:1000, guinea pig anti-rabbit prolactin, obtained from Alfred F. Parlow, NIDDK; 1:500, mouse anti-human TGF-β1; Chemicon Inc., Temecula, CA; 1:500, goat anti-human EGF; R&D Systems Inc.). Membranes were then exposed to the appropriate dye-conjugated secondary antibodies (IRDye 800; Rockland, Gilbertsville, PA): goat anti-guinea pig IgG (1:3000) for prolactin; goat anti-mouse IgG (1:2000) for TGF-β1; and donkey anti-goat IgG (1:2000) for epidermal growth factor [EGF]. Reference samples were: sheep prolactin, 22.5 kDa (Sigma-Aldrich, St. Louis, MO), and human EGF, 6.35 kDa (Oncogene, Cambridge, MA). Near-infrared fluorophores (IRDye-800; Rockland) were determined (Odyssey Infrared Fluorescence Imaging System; LiCor Instruments, Lincoln, NB), and densitometry was conducted on computer (Labworks 4.0; Ultra-Violet Products, Inc., Upland, CA).