Smad2 3 antibody

PHBF cells were plated at 100,000 cells/well in a 24-well cell culture plate overnight, serum-starved for 24 h and then cultured in serum-free DMEM with the indicated concentration of TGFβ1 with or without Galunisertib for 30 min. Cells were lysed in protein lysis buffer containing T-PER tissue protein extraction reagent (ThermoFisher, #78510), cOmplete™ Protease Inhibitor Cocktail (Sigma-Aldrich, #11697498001) and PhosSTOP™ phosphatase inhibitor cocktails (Sigma-Aldrich, #4906845001). Total protein was diluted and normalized to 0.5 µg/µL with 4× LDS sample buffer (ThermoFisher, #84788). In total, 10 µg of total protein was loaded into each well of a NuPAGE 4–12% Bis-Tris Midi Gel (Invitrogen), followed by protein transfer from the gel to the membrane using Trans-Blot Turbo (Bio-Rad). The Phospho-Smad2 was first revealed following the general protocol western blot from Bio-Rad. Briefly, the membrane was blocked for 1 h, incubated with Phospho-Smad2 antibodies overnight at 4 °C (Ser456/467, 1:200, Cell Signaling #3108, clone 138D4), washed and incubated with secondary antibodies goat anti-rabbit HRP-linked (Cell Signaling, #7074, dilution: 1:15,000). To analyse the total Smad2/3, the membrane was stripped and then incubated with Smad2/3 antibodies (Cell Signaling # 8685, clone D7G7, dilution 1:1000).

PLC-8024 cells were treated with rGDF1 at 50 ng/mL or rTGF-β1 at 10 ng/mL or vehicle control for 30 min. The cells were washed twice with cold PBS and cross-linked in 16% formaldehyde (w/v) at room temperature for 10 min. Then, the cells were resuspended in glycine solution (10×) to a final concentration (1×). After washing with cold PBS, cells were collected by centrifugation at 3000 g for 5 min. Cell lysis and MNase digestion were performed using the Agarose chip kit (Thermofisher, US) according to the manufacturer’s standard protocols. Supernatants were incubated with SMAD2/3 antibodies (Cell Signalling Technology, 8685) or isotype control IgG (Cell Signalling Technology, 2729) overnight at 4°C. Protein A/G beads were used to capture antibody-DNA complexes, and the DNA was purified and detected by qPCR analysis. Primers used in chip-qPCR analysis were listed in Supplementary Table 4.

For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA). Anti-β-actin, -NPM1, -Sp1 and -TGF-β antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-E-cadherin, -N-cadherin, -vimentin, -Snail, -Twist and -CUG2 antibodies were obtained from Abcam (Cambridge, MA). For inhibition of protein kinases, wortmannin, PD98059, SP600125, and SB203580 were purchased from Calbiochem (San Diego, CA). TGF-β1 was obtained from R&D Systems (Minneapolis, MN). The TGF-β inhibitor EW-7197 was synthesized as described previously [21 (link)].