Anti cldn1

Immunohistochemical staining of LIN28B/CLDN1/NOTCH3 was performed following standard protocols. Briefly, paraffin-embedded tissue blocks were cut into 4 μm–thick sections. The tissue sections were incubated with anti-CLDN1 (catalog: 71-7800, Invitrogen; dilution 1:1,000), and anti-NOTCH3 (catalog ab23426, Abcam; 1:100) overnight at 4°C; incubated in Envision+ kits (Dako); and visualized using 0.01% 3,3′-diaminobenzidine. The staining intensities varied from 0 (negative), 1+ (weak), 2+ (moderate), to 3+ (strong). The percentage of cells at each staining intensity level was calculated, and H-score was assigned using the following formula; H-score = (1 × % cells 1+) + (2 × % cells 2+) + (3 × % cells 3+). High expression levels were designated as follows: LIN28B ≥ 120, CLDN1 ≥ 200, NOTCH3 ≥ 100. The score of immunohistochemical staining was evaluated independently by 2 investigators.

Anti-CD81 monoclonal antibody (mAb) 5A6 from Santa Cruz Biotechnology (Paso Robles, CA), anti-SRB1, anti-CLDN1, anti-OCLN antibodies, Alexa 488- and horseradish peroxidase (HRP) conjugated anti-goat or anti-rabbit or anti-mouse IgG from Invitrogen (Carlsbad, CA) were used in our study. Anti-NS3 and anti-core antibodies were purchased from Abcam (Toronto, Ontario, Canada).

All cells were lysed in 1% NP40 buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 10% glycerol, 2 mM EDTA) supplemented with 1 mM cOmplete Mini protease inhibitors (Roche) and 1 mM sodium orthovanadate (ThermoFisher). Proteins were separated on a 4–20% SDS-PAGE gel (BioRad) and transferred to polyvinylidene difluoride (PVDF) membrane. Membrane was incubated in 5% BSA and 0.1% Tween-20 in PBS for 1 hr, incubated overnight at 4°C with primary antibodies (1:10000 anti-β-actin, Santa Cruz #sc-47778; 1:1000 anti-SHC, BD Transduction #610878; 1:1000 anti-ERK, Invitrogen #13–6200; 1:1000 anti-phospho-SHC (Tyr239/240), Cell Signaling #2434; 1:2000 anti-phospho-p44/42 ERK (Thr202/Tyr204), Cell Signaling #4370; 1:1000 anti-EGFR, Cell Signaling #2232; 1:700 anti-phospho-Thr/Tyr, Cell Signaling #9381; 1:500 anti-CLDN1, Invitrogen #37–4900; 1:350 anti-OCLN, Invitrogen #33–1500), then incubated for 1 hr at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:10000 anti-rabbit, Cell Signaling #7074; 1:10000 anti-mouse, Cell Signaling #7076). Membrane was added SuperSignal West Pico PLUS Chemiluminescent or West Femto Maximum Sensitivity substrate (ThermoFisher) and exposed to CL-XPosure film (ThermoFisher).