Anti yap

Muscles were lysed in modified RIPA buffer (#C500005, Sangon Biotech) and protease inhibitors, including 1 mm phenylmethylsulfonyl fluoride, 1 μg/μl pepstatin, 1 μg/μl leupeptin and 2 μg/ml aprotinin. After centrifugation at 10000 RPM at 4 °C, the supernatant (lysate) was collected. Protein concentration was measured using a Pierce BCA kit (#23225, Thermo Scientific). Samples (50 μg protein) were resolved by SDS‒PAGE and transferred to a nitrocellulose membrane, which was incubated in 5% milk in phosphate buffered saline (PBS)-0.3% Tween 20(#A600560, Sangon Biotech) overnight at 4 °C and then with the following primary antibodies in 2% milk in PBS-Tween buffer: anti-MuSK (1:1000, sc-33204, Santa Cruz Biotechnology), anti-YAP (1:1000, #4912, Cell Signaling Technology), anti-YAP^ser127 (1:500, #4911, Cell Signaling Technology), anti-Alpha Dystroglycan (1:500, ab106110, Abcam) and anti-GAPDH (1:2000, #437000, Thermo Scientific). After washing, the membrane was incubated with PBS-Tween buffer containing HRP-conjugated goat anti-mouse and anti-rabbit IgG from Pierce (1:2000, PI-32230 (anti-mouse), PI-32260 (anti-rabbit)). The immunoreactive bands were exposed to autoradiography films, and the grayscale was quantified by ImageJ (NIH), as described previously [31 (link), 32 (link)].

After fixation with 4% formalin/phosphate-buffered saline, paraffin-embedded livers were sliced and immunostained using mouse monoclonal antibody anti-proliferating cell nuclear antigen (PCNA) (Agilent, Santa Clara, CA, USA) and anti-YAP (Cell Signaling Technology, Danvers, MA, USA). Staining was developed with diaminobenzidine (DAB), using DAKO Envision System (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Sections were counterstained with hematoxylin. At least 30 high-power fields were counted per slide.

Whole-cell lysates were prepared with RIPA buffer containing protease inhibitor and phosphatase inhibito. The protein concentrations were measured using Bradford. The following antibodies were used: anti-YAP, anti-CTGF, anti-CYR61 and anti-cyclinE from Cell Signaling Technology; anti-GAPDH from Santa Cruz Biotechnology; and anti-NUP37 from Abcam.

Western blots were carried out as previously described48 (link)49 (link). Briefly, 50–100 μg of protein from whole cell extracts from both tumor and non-neoplastic liver were re-suspended in 4X tris-glycine sample buffer, boiled at 95 °C for 5–10 min, and fractionated on a 4–20% tris-glycine gel (Invitrogen, Carlsbad, CA) for 90 min at 125–130 V. Proteins were transferred to PVDF membranes and then blocked with a solution of either 5% BSA or 3% BSA and 5% milk in tris-buffered saline with Tween 20 (TBST) before immunodetection with the following antibodies: anti-YAP (Cell Signaling #4912, Danvers, MA), and anti-GAPDH (Millipore #MAB374, Billerica, MA). Overnight primary antibody incubation was followed by washes (60 min at room temperature) in TBST before incubation in secondary antibody for 1 hour (horseradish peroxidase-conjugated goat anti-rabbit IgG, Cell Signaling; or goat anti-mouse IgG, Bio-Rad, Hercules, CA). After washing with TBST, proteins were visualized using the ECL detection system (NEN, Boston, MA). Coomassie gels were used to ensure equal protein loading. Band densities were analyzed using AlphaEase FC software version 4.1.0 (Alpha Innotech) to determine relative protein expression, and were then normalized to GAPDH band densities.