Anti smad2

IF and IHC were performed according to the conventional protocols. The primary antibodies used for IF were anti-YAP (Abcam, Hong Kong, China #ab52771) and anti-βTrCP (Abcam, #ab233638). The primary antibodies used for IHC were: anti-YAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-101199) and anti-ISG15 (Abcam, #ab233071). For IB, the proteins were resolved on SDS-PAGE gels according to the conventional protocols. The primary antibodies used were anti-ISG15 (Abcam, #ab233071), anti-YAP (Abcam, #ab52771 and Santa Cruz, #sc-101199), anti-GAPDH (CST, #5174 and #51332), anti-Ub (Abcam, #ab7780 and #ab7254), anti-PSMB5 (Abcam, #ab167341), anti-βTrCP (Abcam, #ab71753 and #ab233638), anti-YAP^O241 (developed by Biolynx, Hangzhou, China), anti-YAP^P127 (Abcam, #ab76252), anti-YAP^P397 (CST, Boston, MA, USA, #13619), anti-HA (Abcam, #ab9110 and #ab18181), anti-TEAD4 (Abcam, #ab197589 and #ab58310), anti-6PGL (Abcam, #ab229872), anti-FLAG (CST, #8146 and #2368), anti-UbCH8 (Abcam, #ab177485), anti-HERC5 (Invitrogen, Carbsland, CA, USA, #703675), anti-ATG5 (Abcam, #ab221604), anti-LATS1 (Abcam, #ab243656), anti-CK1 (Abcam, #ab270997 and #ab115293), anti-SMAD2 (Abcam, #ab40855), anti-alpha fetoprotein (AFP, Abcam, #ab284388) and anti-albumin (Alb, Abcam, #ab207327). ELISA kits (Yingxin, Shanghai, China) were used to measure the concentration of YAP protein and Rib-5-P.

Total protein was isolated from cell lysates or tissues by using RIPA buffer. The concentration of protein was detected with a BCA protein kit (Thermo Fisher Scientific). Then, proteins (40 μg per lane) were separated with 10% SDS-PAGE gel and then transferred into polyvinylidene fluoride (PVDF, Thermo Fisher Scientific) membranes. After blocking with 3% skim milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, United States). The primary antibodies used in this study as follows: anti-E-cadherin (Abcam, Cambridge, MA, United States; 1:1000), anti-vimentin (Abcam; 1:1000), anti-α-SMA (Abcam; 1:1000), anti-smad2 (Abcam; 1:1000), anti-smad3 (Abcam; 1:1000), anti-USP4 (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Akt (Abcam; 1:1000), anti-ERK (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.

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.

Paraffin-embedded mouse kidney samples were sliced into 4 μm-thick sections and subjected to IF staining according to an established protocol. Briefly, kidney sections were deparaffinized, followed by antigen retrieval, treatment with peroxidase block, and incubation with mouse anti-E-cadherin (1:200, Cat. No. ab231303, Abcam) and primary antibodies overnight at 4 °C. After washing, sections were incubated with Alexa Fluor 488-labeled anti-mouse secondary antibodies in the dark at room temperature for 1 h. After treatment with an anti-fluorescence quencher containing 4′,6-diamidino-2-phenylindole, the sections were washed and mounted with ProLong Gold (Thermo Fisher Scientific, Shanghai, China). The fluorescence staining area was measured using ImageJ software. After treatment, HK2 cells were fixed as necessary in 4% paraformaldehyde for 20 min, permeabilized, and blocked with 0.1% Triton 100 in 2% BSA for 1 h. HK2 cells were then incubated with anti-SMAD2 (1:200, Abcam, Cat. No. 33875) and subjected to IF staining as previously described.

GBC cells or tissues were isolated from cells by RIPA buffer and quantified by BCA kit (Beyotime). Proteins were separated with SDS-PAGE (10%), and then proteins were transferred onto PVDF (Bio-Rad) membranes. After that, the membranes were incubated with primary antibodies at 4 °C overnight after blocked with 5% skim milk for 1 h. The primary antibodies were as follows: anti-SMAD2 (Abcam, Cambridge, MA, USA; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). Then, the membranes were incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG; Abcam, 1:5000) at room temperature for 1 h. β-actin was used as an internal control. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to visualize the protein bands. ImageJ software (version 2.0; National Institutes of Health) was used to quantify the intensity of the bands.