D7g7 xp

Immunofluorescence analysis of Smad2/3 nuclear translocation was performed in RAW 264.7 cells. The cells were plated on Millicell^® EZ slides (Merck KGaA, Darmstadt, Germany) at a density of 3 × 10⁴ cells/cm². Cells were stimulated with EMD or TGF-β, with and without the SB431542 inhibitor. Cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Smad2/3 (1:800; D7G7 XP^®, Cell Signaling, Danvers, MA, USA, #8685) was applied to the fixed cells overnight at 4 °C. Detection was performed with a goat anti-rabbit Alexa 488 secondary antibody (CS-4412, 1:800, Cell Signaling Technology). We captured the images on a fluorescence microscope with the DAPI-FITC dual excitation filter block (Echo Revolve Fluorescence Microscope, San Diego, CA, USA).

Immunofluorescence analysis of Smad2/3 and p65 nuclear translocation was performed in RAW264.7 cells and gingival fibroblasts, respectively. The cells were plated on Millicell^® EZ slides (Merck KGaA, Darmstadt, Germany) at a density of 15,000 cells/cm². Cells were subjected to 30% lysates from PRF and UBC for one hour following overnight serum starvation. To induce inflammation, the cells were exposed to LPS from Escherichia coli 0111:B41 (Sigma–Aldrich, St. Louis, MO, USA) for 40 min. The cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA) and permeabilized with 0.3% Triton X-100 (Sigma–Aldrich, St. Louis, MO, USA). We used Smad2/3 (1:800; D7G7 XP^®, Cell Signaling, MA, USA, #8685) and NF-κB p65 antibodies (IgG, 1:400, Cell Signaling Technology, #8242) at 4 °C overnight. Detection was performed with a goat anti-rabbit Alexa 488 secondary antibody (CS-4412, 1:800, Cell Signaling Technology). We captured the images on a fluorescence microscope with the DAPI-FITC dual excitation filter block (Echo Revolve fluorescence microscope, San Diego, CA, USA).