Amersham ecl prime western blotting detection system

Tumor cell lines (1 × 10⁶) were washed in phosphate-buffered saline (PBS) and lysed in LDS sample buffer (Invitrogen, Thermo Fisher Scientific). The cell lysate was subjected to electrophoresis in a 4–12% NuPAGE Bis–Tris SDS-PAGE gel (Invitrogen, Thermo Fisher Scientific) under reducing condition and transferred to an Immobilon-P membrane (Merck Millipore). The membrane was blocked in PBS containing 0.01% Tween 20 and 5% non-fat dry milk for 1 h at room temperature and incubated with polyclonal rabbit anti-human PD-L1 (E1L3N, Cell Signaling Technology) diluted 1:1000 in blocking buffer for overnight at 4 °C, or anti-β-actin mAb (C4, Santa Cruz Biotechnology) diluted 1:2000 in blocking buffer as the control for 1 h at room temperature. After washing, the membrane was incubated with horseradish peroxidase-labeled sheep anti-rabbit or anti-mouse IgG and visualized using the Amersham ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences).

Proteins were extracted from Epstein-Barr virus (EBV)-transformed lymphoblastoid cells with RIPA buffer (Sigma-Aldrich) supplemented with proteinase inhibitor (Roche). Twenty micrograms of protein were loaded into a 10% Tris-HCl gel (Bio-Rad). A mouse mAb against N-terminal TET2 (1:500 dilution, hT2H21F11, #MABE462; Merck, Kenilworth, NJ, USA) and rabbit polyclonal antibody against vinculin (1:1000 dilution, H-300, sc-5573; Santa Cruz Biotechnology, Dallas, TX, USA) was used, at 4 °C overnight incubation. Secondary antibodies against mouse and rabbit were incubated at RT for 2 h. Proteins were visualized by Amersham™ ECL Prime Western Blotting Detection System (GE Healthcare). Immunoblots were quantified with ImageJ software (http://imagej.nih.gov/ij/). Full gel image of an extended TET2 western blot run with two different amount of protein extract is shown in Supplementary Fig. 3a.

Primary human arterial endothelial cells and PASMC were lysed in RIPA buffer (Sigma-Aldrich) and 10 μg of protein were run on a 10% SDS polyacrylamide gel, followed by electrotransfer to an Amersham Hybond P 0.45 PVDF Blotting Membrane (GE Healthcare, Wien, Austria). After blocking with 5% non-fat dry milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20), the membrane was incubated overnight at 4°C with one of the following antibodies: anti-p38, anti–phospho-p38 (T180/Y182), anti-c-Jun; anti-phospho c-Jun (S73), anti-phospho Erk1/2 (T202/Y204), anti-Erk1/2, anti-phospho c-Fos (S32) all from Cell Signaling and anti-c-Fos (Novus Biologicals, Cambridge, UK) were used at dilution of 1:1000; and anti-TLR4 (Abcam), anti-RAGE (1:500; Abcam) and rabbit anti–alpha-tubulin (1:5000; Cell Signaling, Boston, MA, USA). After washing, the membranes were incubated with horseradish-peroxidase–labelled secondary antibodies (1:5000, anti-rabbit-HRP, Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and proteins detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare). Antibodies were removed by incubating the membrane for 15 min. with stripping buffer (RestoreTM PLUS Western Blot Stripping Buffer; Thermo Scientific). Densitometric analysis was performed with ImageJ (National Institutes of Health, USA).

Protein lysate from 1 x 10⁶ cells were extracted on ice using Golden Lysis Buffer (10 mM Tris pH 8.0, 400 mM NaCl, 1% Triton X-100, 10% Glycerol+Complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Sigma)). Protein concentration was measured using a BCA Protein Assay Kit (Pierce) and analyzed on the Versamax spectrophotometer at a wavelength of 560 nm. Appropriate volumes containing 20 μg of protein lysates combined with NuPage LDS Sample Buffer and NuPage Reducing Agent (Invitrogen) were run on 4–12% (or otherwise indicated) NuPage Bis-Tris gels in MOPS buffer. Proteins were transferred onto a PVDF membrane (Millipore), blocked in 5% milk (TBST + dry milk) for 1 hour and incubated in the primary antibody (in 5% milk) overnight at 4°C. Membranes were washed with 0.05% TBST (TBS + 5% Tween) and secondary antibody incubations were done at room temperature for 1 hour. Proteins were visualized using Amersham ECL Prime Western Blotting Detection System and Amersham Hyperfilm ECL (GE Healthcare).
The following primary antibodies were used: mouse anti-Actin (1:10,000; Abcam), mouse anti-myc-tag (1:1000; Cell Signaling Technology), mouse anti-KRAS (1:1,000; Sigma). Secondary antibodies, including goat anti-rabbit (Santa Cruz) and goat-anti-mouse (GE Healthcare), were used at a concentration of 1:10,000.

For dot-blot analyses, purified MuSK protein was diluted with PBS to make solutions of 1∶10, 1∶100, and 1∶1000 concentration, and 2 µl of each dilution was applied directly to nitrocellulose paper, which was then incubated overnight with a 1∶500 dilution of serum sample from mice injected with MuSK or control vehicle. The nitrocellulose paper was then blocked with 2% BSA for 2 hrs, incubated with anti-mouse HRP-labeled secondary antibody, and analyzed with an Amersham ECL Prime Western blotting detection system (GE Health Care). For Western blot analysis primary antibodies against MuSK (Abcam, USA) and the AChR γ subunit (Santa Cruz Biotechnology, USA) were used, according to manufacturers' instructions.

Protein concentrations of cell lysates were determined using the Thermo Scientific Pierce (Waltham, MA, USA) BCA Protein Assay Kit according to manufacturer’s instructions. The following antibodies were used: monoclonal mouse anti-human cMYC (1:200, catalog no. sc-40; Santa Cruz, Dallas, TX, USA), monoclonal mouse anti-human HDAC2 (1:1000; catalog no. sc-81599; Santa Cruz), polyclonal rabbit anti-human AcH4 (1:1000; catalog no. 06-866; Milipore, Billerica, MA, USA) and mouse monoclonal anti–β-actin (1:10000; catalog no. A5441; Sigma-Aldrich) and detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) on PVDF membrane with Chemi-Smart 5000 Technology (Vilber Lourmat, Eberhardzell, Germany). Uncropped images were contrast enhanced with Chemi-Capt 5000 (Vilber Lourmat) and subsequently cropped in Microsoft Office PowerPoint 2007 SP3 (Microsoft Corporation, Redmond, WA, USA).