Anti phospho smad2

The expression levels of proteins in livers and mouse HSCs were analyzed as previously described [35 (link)]. Primary antibodies were as follows: anti-mouse Cav1 (Cat. No.610406, 1:1000, BD Biosciences), anti-mouse α-SMA (Cat. No.A5228, 1:1000, Sigma-Alorich; Shanghai, China), anti-mouse Collagen α1(I) (Cat. No.ab6308, 1:200, Abcam), anti-mouse Collagen α1(III) (Cat. No.ab7778, 1:200, Abcam), anti phospho-Smad2 (Cat. No.ab53100, 1:1000, Abcam), anti-Smad2 (Cat. No.ab33875, 1:1000, Abcam), anti-β-Actin (Cat. No.12262, 1:1000, Cell Signaling, Shanghai, China). Goat anti-mouse IgG labeled with HRP (Cat. No.ab6789, 1:2000, Abcam) was used as secondary antibodies.

We performed Western blotting, immunoprecipitation, and immunofluorescence analysis, as previously described [32 (link)]. Antibodies obtained from the following sources: Anti-phospho-SMAD2, anti-phospho-SMAD3, anti-SMAD2, anti-SMAD3, anti-SMAD4, TβRI, TβRII, and TrkB were from Abcam. anti-Flag and β-actin were from Sigma-Aldrich; anti-V5 was from Invitrogen; anti-Myc, α-tubulin, and lamin were from Santa Cruz Biotechnology.

Protein expression was studied on purified cell lysates and concentrated conditioned media. Cell total proteins were extracted using the T-PER Tissue Protein Extraction Reagent (ThermoFisher Scientific) with the Halt Protease & Phosphatase Inhibitor (ThermoFisher Scientific). Proteins were separated in 4–20% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA). Membranes were incubated with the following antibodies: human primary anti-Notch cleaved 1 (1:1000, Cell Signaling Technology, Pero, Italy); purified human anti-HES1 (1:1000, Cell Signaling Technology); anti-TGFβ (1:500 R&D); anti-phosphoSMAD2 (1:1000, Abcam, Cambridge, UK), anti-SMAD2/3 (1:1000, Cell Signaling Technology) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). In addition, a secondary anti-rabbit (1:2000, Cell Signaling Technology) or anti-mouse (1:2000, Biorad Laboratories, Hercules, CA, USA) antibody was used. The chemiluminescence signal from proteins was revealed using Clarity Max Western ECL Substrate (Bio-Rad) and captured with the ChemiDoc MP instrument (Bio-Rad Laboratories) using Image Lab 5.2.1. The relative density of the bands was calculated using the ImageLab software.