Comprehensive Molecular Profiling of Parotid Tissue

Total RNA extraction, reverse transcription, and quantitative PCR (qPCR) were performed as reported [44 (link),45 (link)]. Primers for swine GAPDH, C1qA, TNF-α, IFN-γ, IL-4, IL-6, P53, AIF1, ADGRE 1, ITGAX, HGF, and ARG 1 were designed using Primer3 software (http://bioinfo.ut.ee/primer3/) and listed in Supplementary Table S1. For protein extraction, fresh parotid samples were homogenized in T-PER reagent containing protease inhibitors (Pierce, WA, USA). The concentration of protein was quantified using BCA, and the loading quantity was 25 μg per lane. Western blotting was done using primary antibodies against AQP5 (1:5000, ab78486), Ptch1 (1:5000, ab51983), TNF-α (1:5000, ab1793), IL-6 (1:200, ab6672), P53 (1:500, ab154036) (these 5 antibodies are from Abcam, Boston, MA, USA), Gli1 (1:1000, bs-1206R), F4/80 (1:1000, bs-7058R), AIF1 (1:1000, bs-1363R), and VEGF (1:100, bs-1313R) (these 4 antibodies are from Bioss, Woburn, MA, USA). All PCR and Western blot analyses were repeated at least three times.