Anti hb egf

Antibodies against phospho-EGFR (Tyr1068), EGFR and MUC5AC (45 M1) were obtained from Abcam (Cambridge, MA, USA). Antibodies against phospho-ERK (Thr202/Tyr204) and ERK, and PD98059 were from Cell Signaling Technology (Danvers, MA, USA). The anti-EGFR neutralizing antibody was from Chemicon (Temecula, CA, USA). The other neutralizing antibodies (anti-TGF-α, anti-AR, and anti-HB-EGF), species- and isotype-matched control antibodies and recombinant AR were from R&D Systems, Inc. (Minneapolis, MN, USA). The GAPDH antibody was from TDYbio (Beijing, China). The MUC5B antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AG1478, GM6001, TAPI-1 and actinomycin D were purchased from Calbiochem (San Diego, CA, USA). Unless stated otherwise, all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Male 8–10 weeks old C57BL/6 mice were purchased from Shanghai SLAC Co. Ltd. (Shanghai, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine (No. SYKX-2008-0050). For intraperitoneal (i.p.) transplantation of microcapsules, the mice were randomly divided into four groups with eight mice per group. The groups were as follows: SHAM (blank control, without administration of LPS and D-gal), CON (control, APA), HEP (hepatocytes, 5 × 10⁶), UMSC-HEP (UMSCs 2 × 10⁶ and hepatocytes 5 × 10⁶ cells), and HNF4α-UMSC-HEP (HNF4α-UMSC 2 × 10⁶ and hepatocytes5 × 10⁶ cells). ALF was induced by i.p. injection of the combination of LPS (50 μg/kg) and D-gal (800 mg/kg) dissolved in PBS for 24 h after microcapsule transplantation. The HB-EGF-neutralizing antibody (anti-HB-EGF, R&D, MN, USA) and control IgG (AB-108-C, R&D, MN, USA) were reconstituted in sterile PBS and administered to ALF mice (10 μg/injection, i.p.).
The enzyme activities of aspartate aminotransferase (AST) and alanine transaminase (ALT) were measured using a standard clinical automatic analyzer (Dimension Xpand; Siemens Dade Behring, Munich, Germany). The levels of TNF-α, CXCL15 (a homolog of human IL-8), and LDH were detected by ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.

Anti-RAS monoclonal antibody recognizing G-domain of all major RAS isoforms was purified from a hybridoma cell line provided by FNLCR and used at 1:2000 dilution as previously described^{18 (link)}. Other commercially available primary antibodies used were: anti–Phospho-p44/42 MAPK (phosphorylated ERK1/2, Thr202/Tyr204, Cell Signaling Technology #4377), anti-p44/42 MAPK (ERK1/2, Cell Signaling Technology #4696), anti-HB-EGF (R&D Systems, #AF-259-NA;), anti-p27^Kip1 XP (Cell Signaling Technology #3686), Phospho-RB (Ser807/Ser811, Cell Signaling Technology #8516), anti-p21^WAF/Cip1 (Cell Signaling Technology #2947T), anti-α-Tubulin (Cell Signaling Technology #2144), and anti-vinculin (Cell Signaling Technology #13901). Fluorescently-labeled secondary antibodies obtained from LI-COR Biosciences and used at 1:10,000 dilution were: IRDye 680RD goat anti-mouse (926-68070), IRDye 800CW goat anti-rabbit (925-322211), and IRDye 800CW donkey anti-goat (925-32214). Western blot images were acquired using an Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified by densitometry using NIH ImageJ software.