Smad2 3 antibody d7g7 xp rabbit mab 8685

Gingival fibroblasts seeded onto Millicell EZ slides (Merck KGaA, Darmstadt, Germany) were serum-starved overnight before being exposed to the indicated cell lysates with and without SB431542 for 30 min. Then, paraformaldehyde fixation blocking was conducted with 5% bovine serum albumin and 0.3% Triton X-100 in PBS. After permeabilization with 0.1% Triton X-100, cells were incubated with Smad2/3 antibody (D7G7 XP rabbit mAb #8685, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. For detection, the Alexa Fluor 488-conjugated antibody (Cell Signaling Technology) was used. Images were captured with a fluorescent microscope (Axio Imager M2, Carl Zeiss AG, Oberkochen, Germany).

Gingival fibroblasts were plated in growth medium onto Millicell^® EZ slides (Merck KGaA, Darmstadt, Germany). The following day, cells were treated with serum-free medium overnight. The next day, cells were exposed to 30% of supernatants (SC and SF) with and without SB431542 for 30 min. Cells were then fixed in paraformaldehyde and blocked in 5% bovine serum albumin (BSA) and 0.3% Triton X-100 in phosphate-buffered saline (PBS) at room temperature, after which permeabilization with 0.1% Triton X-100 took place. Cells were incubated with Smad2/3 antibody (D7G7 XP^® rabbit mAb #8685, Cell Signaling, MA) overnight at 4 °C. Then, an Alexa Fluor^® 488-conjugated secondary antibody (Anti-Rabbit, Cell signaling Technology, Danvers, MA, USA) for 1 h at room temperature was used. Finally, cells were washed and mounted onto glass slides. Images were captured under a fluorescent microscope (Axio Imager M2, Carl Zeiss AG, Oberkochen, Germany).