Super rx fuji x ray film

Whole cellular protein extracts were prepared in 95°C Laemmli sample buffer and mechanically sheared by pressing few times through syringes (26 G). Protein concentration was determined using a Pierce™ 660 nm Protein Assay (Thermo Scientific). A total of 5 µg protein per extract was separated on denaturing 10–20% gradient SDS-PAGE gels. Proteins were transferred on PVDF transfer membranes (0.45 µm, Perkin Elmer). Membranes were probed with the following primary antibodies: anti-calretinin (Sigma, HPA007306), mouse anti-actin (#69100) from MP Biomedicals, N-Cadherin (BD Biosciences, 610920), YAP (Cell Signaling 4912), Mesothelin (Rockland Inc. 200-301-A88), GATA4 (C-20) (Santa Cruz sc-1237), p62 (Progen GP62-C), LC3B (Cell Signaling 2775S), p53 (DO-1) (Santa Cruz sc-126), Thy1 (H-110) (Santa Cruz sc-9163), and γ-H2AX (Millipore 05-636). Membranes were then incubated with the secondary antibody rabbit anti-mouse IgG-HRP (A-5420) from Antell, and goat anti-rabbit IgG-HRP (#7074) from Cell Signaling. The signals were detected by enhanced chemiluminescence (ECL™ Western Blotting Reagents, GE Healthcare) and detected on photosensitive film (Super RX Fuji x-Ray Film, Fujifilm). Proteins were quantitated with densitometry using Image J software (Version 1.42q, USA).

Total protein extracts were prepared by lysing the cells with hot Laemmli sample buffer (60 mM Tris-Cl pH 6.8, 100 mM DTT, 5% glycerol, 1, 7% SDS) and pressed few times through syringes (26 G). Protein concentration was determined using a Pierce^™ 660nm Protein Assay (Thermo Scientific). A total of 5 μg protein per extract was separated on denaturing 10–20% gradient SDS-PAGE gels. Proteins were transferred on PVDF transfer membranes (0.45 μm, Perkin Elmer). For Western blotting, membranes were probed with the following primary antibodies: anti-calretinin (Sigma, HPA007306), anti-cyclin E (Santa Cruz Sc-247), anti-cyclin A (Millipore, 06–138), anti-NRF-1 (Santa Cruz SC33771, H300) and mouse anti-actin (#69100) from MP Biomedicals. Membranes were then incubated with the secondary antibody rabbit anti-mouse IgG-HRP (A-5420) from Antell, and goat anti-rabbit IgG-HRP (#7074) from Cell Signaling. The signals were detected by enhanced chemiluminescence (ECL^™ Western Blotting Reagents, GE Healthcare) and detected on photosensitive film (Super RX Fuji x-Ray Film, Fujifilm). Relative quantification was assessed using ImageJ (NIH).