Dsred

Total proteins were extracted in sodium dodecyl sulfate utilizing buffer (SUB) at different time points (containing 8M urea, 2% β-mercaptoethanol, and 0.5% SDS) and electrophoresed in 12% SDS-PAGE gels. Proteins were blotted on nitrocellulose membranes and probed with specific antibodies against PABPN1 (1:2,000) (Abcam, USA), Dsred (1:20) (Biovision, USA), and myosin (1:200) (DSHB, Iowa City, IA, USA). Parallel samples were probed using monoclonal actin antibody (Chemicon International, USA) to confirm the equal loading of lysates between lanes. After incubation with specific secondary horseradish peroxidase (HRP)-conjugated antibodies, the membranes were revealed using the western blot chemiluminescence reagent plus kit (NEN Life Sciences Products, Boston, MA, USA).

Mouse salivary glands were 4% formaldehyde fixed (24 hr at room temperature) and processed for paraffin embedding. Following dehydration, the tissue was embedded in paraffin and sliced into 5-μm sections. The sections were dewaxed, boiled for 8 min in pre-heated 10 mM citric acid retrieval buffer (pH 6.0) containing 0.05% Tween 20, washed thoroughly prior to primary antibody exposure, and labeled for the following markers: EpCAM (1:100; Schnell et al., 2013 (link)), β-catenin (1:100; Transduction Laboratories, 610154), and dsRed (1:100; BioVision, #3984-100). For fluorescence microscopy, Alexa Fluor 488 goat anti-rabbit (Life Technologies, A11008) or Alexa Fluor 594 donkey anti-mouse (Life Technologies, A21203) conjugates at 1:300 dilution were used as secondary antibodies. Nuclear staining was performed with DAPI (Sigma-Aldrich). H&E staining was performed according to standard protocols. Visualization for bright-field microscopy was accomplished by addition of specific secondary biotin-carrying antibodies (Dako), an avidin-biotin-horseradish peroxidase complex (ELITE ABC Kit, Vector Laboratories) and the diaminobenzidine chromogen. Nuclear staining was performed with hematoxylin.