Western blot analysis

The parotid and submandibular gland tissue were homogenized in RIPA lysis buffer and 1 mM PMSF (phenylmethylsulfonyl fluoride) on ice for 30 min, and subsequently centrifuged for 10 min at 10,000 x g and 4˚C in order to extract proteins. The protein contents were determined using nucleic acid protein detector. The protein samples were boiled after adding 4x loading buffer for 10 min at 100˚C and the protein samples of equal quantity were subsequently separated using SDS-PAGE. Proteins with different molecular weights were separated on 10% separation gel and 5% stacking gel. The separated proteins were subsequently transferred onto polyvinylidene difluoride membranes, which were already soaked in methanol for 15 min, and blocked with 5% skim milk prepared with PBST (ratio of PBS and Tween-20 was 2,000:1) for 2 h. The protein blots were subsequently incubated with primary antibodies for sAA (ab139994, Abcam, dilution, 1:1,000), β-actin (20536-1-AP, Proteintech Group, Inc., dilution, 1:5,000) and β1-AR (ab3442, Abcam, dilution, 1:1,000) overnight at 4 ˚C. Proteins were then incubated with the corresponding Horseradish Peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG (E030120-01, EarthOx Life Sciences, dilution, 1:10,000) for 2 h after washing with PBST in triplicate. The protein blots were stained using ECL luminous solution. Due to the difference of expression abundance, the interest and control proteins were run on different membranes. Protein bands were obtained using GeneGnome XQR gel imaging systems (Syngene) and quantified by ImageJ software (National Institutes of Health).