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. 2017 Mar 20;114(14):E2975–E2982. doi: 10.1073/pnas.1611428114

Fig. S2.

Fig. S2.

Characterization of the NG-Smad3 construct. (A) Amino acid sequence of the NG-Smad3 used in the study. The asterisk denotes a stop codon. (B and C) Quantifying the level of NG-Smad3 relative to endogenous Smad3 using quantitative Western blotting. (B) Cell lysate was loaded at different concentrations to find the range where antibody staining is linear. IB, immunoblot. (C) Linear region (blue-shaded area) is where the change in lysate amount is proportional to the change in fluorescent signal. The relative expression of NG-Smad3 to Smad3 was determined by taking the average of two biological replicates. (D) Western blot against phosphorylated Smad3 (pSmad3) in the nuclear fraction. (E) Western blot against Smad3 in the nuclear fractions. In both D and E, cells were collected 1 h after Tgf-β stimulation (2.4 ng/mL). For Western blotting, cells were treated with Tgf-β1 or a carrier, harvested by trypsinization, and then either pelleted and frozen or fractionated into nuclear and cytoplasmic fractions using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific; 78833). Cell lysates were loaded on Bolt 4–12% gradient SDS/PAGE (Invitrogen) and transferred onto nitrocellulose membranes by wet transfer using a standard wet transfer buffer [25 mM Tris, 192 mM glycine, 20% (wt/vol) methanol] for 1 h at 200 mA at 4 °C. Membranes were dried, blocked using Odyssey blocking buffer (LI-COR; 927-50000) for 1 h at room temperature, and incubated with primary antibodies at 4 °C overnight and secondary antibodies for 1 h at room temperature. Imaging and quantification were performed using a LI-COR Odyssey infrared scanner. Primary antibodies were as follows: rabbit–anti-Smad3 (Cell Signaling; C67H9) at a 1:1,000 dilution, rabbit–anti-pSmad3 (Cell Signaling; C25A9) diluted at 1:1000, and mouse-anti–β-actin (Cell Signaling; 8H10D10) diluted at 1:20,000. All primary antibodies were diluted in blocking buffer. Secondary antibodies, goat–anti-rabbit IRDye 680LT (LI-COR; 925-68021), and goat–anti-mouse IgG (H+L) DyLight 800 Conjugate (Cell Signaling; 5257) were diluted at 1:5,000. All secondary antibodies were diluted in blocking buffer + 0.1% Tween-20 + 0.01% SDS.