Monoclonal mouse anti β tubulin

Primary antibodies were: monoclonal rat anti-ZO-1 (R40.76, 1:500 IB, 1:100 IF a gift from Prof. Daniel Goodenough, Harvard Medical School), polyclonal rabbit anti-ZO-1 (Thermo Fischer Scientific, 61–7300, 1:2000 IB) and monoclonal mouse anti-ZO-1 (Thermo Fischer Scientific, 33–9100, 1:2000 IB), rabbit anti-cingulin C532 (1:5000 IB),⁷⁴ monoclonal mouse anti-GFP (Roche Applied Science, 11814460001, 1:2000 IB, 1:200 IF), mouse anti-myc 9E10 (1:200, IF), guinea pig anti-PLEKHA7 GP2737 (1:300 IF), monoclonal mouse anti-β-tubulin (Invitrogen, 32–2600, 1:5000 IB). The polyclonal rabbit anti-ZO-1 R3 was obtained by immunizing rabbits with a peptide (CRDNSILPPLDKEKGETLLSPLV) corresponding to residues 1673–1695 within the ZU5 domain of mouse ZO-1 (Covalab, Lyon, France), and was used at a dilution of 1:1000 for IB, and 1:100 for IF. Secondary antibodies for IF were anti-mouse and anti-rabbit Alexa Fluor 488, anti-mouse, anti-rabbit, and anti-rat Cy3, anti-guinea pig Alexa Fluor 647 (Jackson ImmunoResearch Europe, Newmarket, UK, 1:300). Anti-mouse and anti-rabbit (1:20000, Promega, W402B and W401B, respectively), and anti-rat (1:10000, Thermo Fisher Scientific, 62–9520) IgG, HRP-conjugated antibodies were used for immunoblotting.

Human HCC cells SK-Hep1, HepG2, and Hep3B were purchased from the ATCC (Manassas, VA) and cultured in Eagle's Minimum Essential Medium (EMEM). All cells were cultured at 37 °C in 5% CO₂, and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Western blot was performed using a 1:1000 dilution of primary polyclonal rabbit anti-EGFR antibody (#2232S, Cell Signaling Technology) per manufacturer instructions. Loading was controlled with a 1:500 dilution of monoclonal mouse anti-β-tubulin (#32-2600, Invitrogen). SK-Hep1 cells were diluted in growth factor reduced (GFR) Matrigel Matrix (Corning), and injected into one flank of female (to avoid male dominance within a cage) nude athymic mice (nu/nu, Jackson Laboratory) at 4–6 weeks of age with weight between 20 and 25 g. ∼5 × 10⁶ cells were implanted per mouse. Anesthesia was induced and maintained via a nose cone with inhaled isoflurane mixed with oxygen at a concentration of 2–4% at a flow rate of ∼0.5 L/min for all in vivo animal experiments.