TGF-β Signaling Modulation in 3T3 Cells

3T3 cells were incubated in a 6-well plate at a density of 1 × 10⁵ cells per well and treated overnight with TGF-β (20 ng/ml), TGF-β (20 ng/ml) plus SB-431542 (MCE, USA), a kind of TGF-β inhibitor, different 4T1 cultured medium after treatment with PBS (4T1-CM), DTX (4T1@DTX), C-Lp/DTX (4T1@C-Lp/DTX), Nanoxel-PM (4T1@Nanoxel-PM), Rg3 (4T1@Rg3), Rg3-Lp (4T1@Rg3-Lp), Rg3/DTX (4T1@Rg3/DTX) or Rg3-Lp/DTX (4T1@Rg3-Lp/DTX) (DTX 0.5 μg/mL). The protein of the cells was then harvested according to the procedures described previously [32 (link)]. 50 mg of protein per lane were loaded on the polyacrylamide gel and then transferred onto a PVDF membrane. The PVDF membrane was incubated with anti-α-SMA (ab124964, Abcam), anti-phospho-Smad2/3 (ab272332, Abcam) and anti-GAPDH (30202ES40, Yeasen) overnight at 4℃ respectively. The following procedure was performed as previously described [32 (link)].