D6694

Dental tissues were obtained for immunohistochemical analysis by ventricular perfusion of 4% PFA. Fixed hemimandibles were decalcified (10% EDTA, 2 weeks), embedded in paraffin, and sectioned (5 μm thick). Immunofluorescence was performed as reported [16 (link)] using the anti-DSCR1 antibody (D6694, SIGMA) using the biotin-labeled anti-rabbit IgG (1:500 dilution, Vector Laboratories), and detection was carried out using Streptavidin Alexa Fluor 488 (1:800 dilution, Life Technologies). Samples were embedded using Fluoromount mounting medium (Novus) containing DAPI (Thermo Fisher Scientific). Images were taken using a Leica TCS SP5 II confocal microscope.

Cells were seeded on cell climbing sheets, fixed with 4% paraformaldehyde. After blocking with QuickBlock™ Blocking Buffer (P0260, Beyotime), cells were given primary antibodies to incubate, including RCAN1 (1:200, D6694, Sigma-Aldrich), YY1 (1:200, ab109228, Abcam), and NFATc1 (1:100, 66963-1-Ig, Proteintech). On the second day, Alexa Fluor 488 goat anti-mouse IgG (H + L) or Alexa Fluor 594 goat anti-rabbit IgG (H + L) secondary antibodies were employed to incubate cells, followed by nuclear staining (0100-20, SouthernBiotech) for 15 min. The fluorescence signals were analyzed by the thunder imager fast high-resolution inverted fluorescence imaging system (THUNDER DMi8, Leica, German).

To prepare aortic protein lysates, mouse aortic samples were isolated, frozen in liquid nitrogen, and then homogenized using a mortar and an automatic bead homogenizer (MagNA lyzer, Roche). Protein extracts were obtained in ice-cold RIPA buffer (50 mM NaCl, 50 mM Tris HCl pH8, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with protease, phosphatase, and kinase inhibitors. Cultured cells were washed with ice-cold PBS and lysed in RIPA buffer.
Immunoprecipitated proteins and protein lysates were separated under reducing conditions on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Proteins were detected with the following primary antibodies: rabbit anti-Rcan1 (1:1000; D6694, Sigma), anti-p-MLC (1:1000; #3671, Cell Signaling), mouse monoclonal anti-β-catenin (1:1000; 610153, BD Biosciences), mouse monoclonal anti-GSK3β (1:1000; 610202, BD Biosciences), mouse monoclonal anti-alpha tubulin (1:40,000; T 6074 Sigma-Aldrich), and anti-GAPDH (1:5000; ab8245 Abcam). HRP-conjugated secondary antibodies were detected with enhanced chemiluminescence (ECL) detection reagent (Millipore).