Immunoblotting of Submandibular Gland and Saliva

Immunoblotting was performed as described previously [6 (link)] using submandibular gland tissue homogenates and saliva. Briefly, submandibular glands were homogenized in a radioimmunoprecipitation assay lysis buffer and were quantified for total protein using a Bio Rad Protein assay buffer (Bio-Rad, Hercules, CA, USA). About 30 µg of total protein extract was separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE; BioRad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride membrane (PVDF). Later, membranes were probed with indicated primary antibodies and incubated again with the secondary antibody. Post incubation blots were developed using a chemiluminescence detection system. For Coomassie Brilliant Blue R-250 staining (CBB), samples were stained with CBB and de-stained using a 30% methanol/10% acetic acid solution.