Amersham biosciences 600 imager

Primary lung fibroblasts were grown to 80% confluency and then treated with cycloheximide (CHX; 50 μg/mL) for 0, 8, 16 and 24 h in the presence or absence of ThG or OSMI‐1. Protein extraction of whole cell lysates were prepared in 2× SDS reducing buffer with protease inhibitors. The protein concentration was measured by a Micro BCA Protein Assay kit (Thermo Scientific). Lysates were then subjected to SDS‐PAGE and western immunoblotting with antibodies against EZH2 (Cell signalling, Cat# 5246), or β‐actin for loading control. The blots were imaged with an Amersham Biosciences 600 Imager (GE Healthcare) and densitometry analysis was done using ImageJ software.

Cells were lysed in the radioimmune precipitation assay (RIPA) buffer (Sigma‐Aldrich) followed by addition of protease and phosphatase inhibitors. The total protein concentration of the lysates was quantitated using a Micro BCA Protein Assay kit (Pierce). Further RIPA was added to equalize the concentrations of the lysates, and 10× reducing agent along with 4× loading buffer was added to the lysates, followed by incubation at 95°C for 5 min. The samples were then subjected to SDS‐PAGE, and Western immunoblotting was performed as described previously (Desai et al., 2014 (link)). Immunoblots were imaged using Amersham Biosciences 600 Imager (GE Healthcare) at UAB and Biorad ChemiDoc XRS+ imager at University of Colorado. Quantification (densitometry) was performed using Fiji software.

Whole cell lysates were collected and quantified with a Micro BCA Protein Assay kit (Thermo Scientific). Nuclear extracts were collected with EpiQuick Nuclear Extraction kit (Epigentek, Farmingdale, NY), then quantified. Lysates or nuclear extracts were subjected to SDS-PAGE and western immunoblotting (WB) as described before 28 (link). The following antibodies were used: Anti-Mof (A300-992A) from Bethyl Laboratories (Montgomery, TX); anti-α-smooth muscle actin (03-61001) from ARP, anti-Nox4 (AF8158) from R&D systems (Minneapolies, MN), anti-Col1A1(cat# GTX82720) from Genetex (Irvine, CA); anti-survivin (#2808), anti-β-actin (#2128), and anti-H3 (#9715) were from Cell signaling (Beverly, MA), anti-H4K16Ac(#61529) and anti-H4K20Me3 (#39671) were from Active Motif (Carlsbad, CA). Immunoblots were imaged with an Amersham Biosciences 600 Imager (GE Healthcare). Densitometry analysis was done using Image J software.

Equal amounts of proteins were fractionated on 4%–20% polyacrylamide gels (GenScript, M42015) or 4%–12% BisTris polyacrylamide gels (Life Technologies, NW04122BOX) and transferred to a polyvinylidene difluoride membrane (Millipore/Sigma, IPVH00010) or nitrocellulose membrane (Bio-Rad, 1620112). When using HRP-conjugated antibodies, the membrane was blocked for 1 h at room temperature in PBS-Tween 20 (PBS-T) plus 5% (w/v) milk and then incubated overnight at 4 °C with appropriate dilutions of the primary antibodies. The membrane was then washed in PBS-T, followed by incubation for 1 h at room temperature in the presence of HRP-conjugated secondary antibodies (1:3000 dilution). After washes in PBS-T, the membrane was developed with enhanced chemiluminescence reagents (Thermo Fisher Scientific, PI32106), and the signal was captured using the Amersham Biosciences Imager 600 (GE Healthcare).
When using LI-COR reagents, membranes were blocked with LI-COR blocking solution and incubated with primary antibodies overnight, followed by three washes in LI-COR blocking solution and incubation with secondary antibodies (1:10,000 dilution) for 1 h in the dark. After three final washes, the membranes were imaged on a LI-COR fluorescent imaging station. Band intensities were quantified using ImageJ software.

The cells were washed with phosphate-buffered saline (PBS) and resuspended in 50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Nonidet P-40 (radioimmune precipitation assay buffer), and protease inhibitors. After ultrasonication using Bioruptor (UCD-250, Cosmo Bio) for 12.5 min (10 s on, 20 s off) at the middle level of output (250 W) at 4 °C, the soluble cellular extracts were recovered after centrifugation for 10 min at 16,000 × g. The protein concentration of each sample was determined using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific), and cell extracts were subjected to WB analysis. The blots were probed with the primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. The immune complexes were visualized using Pierce^TM Western blotting Substrate Plus (Thermo Fisher Scientific). WB results were documented and quantified using ImageQuant LAS 4000 mini and Amersham Biosciences Imager 600 (GE Healthcare).

Immunoblotting was performed essentially as described previously (45 (link)). Briefly, proteins were separated by SDS-PAGE on 12% BisTris gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Invitrogen), and incubated with anti-His₆ (Bethyl Laboratories, Inc.), anti-β-lactamase (Abcam), or anti-GroEL (Sigma) primary antibodies followed by an appropriate horseradish peroxidase-conjugated secondary antibody (Sigma). Chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) on an Amersham Biosciences Imager 600 (GE Healthcare).

Total proteins were isolated from the tissues of adult females, and quantified with a BCA protein assay kit (Applygen Technologies) as described previously [59 (link)]. Western blots were performed using the primary antibodies against β-Cat, Par3, aPKC, Par6, p-Par3, p-aPKC (Invitrogen), and VgA [60 (link)], the corresponding HRP-conjugated secondary antibodies (CWBIO) and an enhanced chemiluminescent reagent (CWBIO). Application of β-actin was used as the loading control. Bands were visualized by an Amersham Biosciences Imager 600 (GE Healthcare) and quantified by ImageJ. For immunoprecipitation, protein extracts were precleared with Protein A-agarose (Sigma-Aldrich) for 1 h at 4°C and incubated with the antibody of β-Cat at 4°C overnight. The immunocomplexes were captured with Protein A agarose (Sigma-Aldrich) and eluted in Laemmli sample buffer, followed by Western blot with antibodies.