D7g7 xp rabbit mab 8685

Gingival fibroblasts were plated in growth medium onto Millicell^® EZ slides (Merck KGaA, Darmstadt, Germany). The following day, cells were switched to serum-free medium and either immediately exposed to the supernatant of the membrane for 30 min (Smad/2/3) or after an overnight starvation in serum-free medium (p-Smad3). Cells were then fixed in paraformaldehyde and blocked in 5% BSA and 0.3% Triton X in PBS at room temperature then permeabilization with 0.1% Triton X was performed. Following this, cells were incubated with p-Smad3 Ser423/425 (ab52903 rabbit, Abcam, Cambridge, UK) and with Smad2/3 antibody (D7G7 XP^® rabbit mAb #8685, Cell Signaling, MA, USA) for overnight at 4 °C. Following an Alexa Fluor^® 488-conjugated secondary antibody (1:1000; Anti-Rabbit, Cell signaling Technology, Danvers, MA, USA) was added for 1 h at room temperature. Cells were washed and mounted onto glass slides. Images were captured under a fluorescent microscope (Axio Imager M2, Carl Zeiss AG, Oberkochen, Germany).

In line with our previous research^{21 (link)}, immunofluorescent analysis was conducted on human gingival fibroblasts plated onto Millicell EZ slides (Merck KGaA, Darmstadt, Germany) stimulated with ADL 10% for 30 min. Cells were fixed using paraformaldehyde and blocked in 5% BSA and 0.3% Triton in PBS at room temperature for 1 h. Cells were next incubated with Smad2/3 antibody (1:800; D7G7 XP Rabbit mAb #8685, Cell Signaling Technology, MA, USA) overnight at 4 °C. Alexa Fluor 488 secondary antibody (1:1000; Anti-Rabbit, Cell Signaling) was added for 1 h. Cells were rinsed and put onto glass slides. Fluorescent images were taken at 4 × by a Zeiss Axiovert 200 M fluorescent microscope.