Imagequant las 4000 scanner

The tissues and cell protein were obtained as previously described [1 (link),17 (link)]. The protein concentration was determined with a Bio-Rad Bradford kit (Bio-Rad Laboratories, Hercules, CA, USA). The samples were boiled for 5 min, and equal volumes were loaded on a sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved proteins were transferred onto a nitrocellulose membrane (Millipore Corporation, Bedford, MA, USA) and probed with type-I collagen (#ab34710, Abcam, Cambridge, UK), fibronectin (#sc71113, Santa Cruz, CA, USA), PAI-1 (#sc8979, Santa Cruz), E-cadherin (#3195, Cell signaling), α-SMA (#48938, Cell signaling), vimentin (#5741, Cell signaling), SMAD3 (#9523, Cell signaling), pSMAD3 (#9520, Cell signaling), STAT3 (#9139, Cell signaling), pSTAT3 (#9145, Cell signaling), and β-Actin (#4970, Cell signaling) followed by a secondary antibody conjugated to horseradish peroxidase and detected with enhanced chemiluminescence reagents (Amersham Bioscience, Buckinghamshire, UK). The luminescent signals were analyzed using an ImageQuant LAS 4000 Scanner of GE Healthcare (Piscataway, NJ, USA).

DIG Gel Shift Kit (Roche, Mannheim, Germany) was performed to detect Smad binding element (SBE) DNA-binding and Smad4 antibody-binding activity, with the instructions of manufacturer. The binding activity of Smad 3/4 in nuclear extract of 8505C was confirmed by EMSA or supershift assay with a DIG-labeled oligonucleotide probe forward 5’-AGTATGTCTAGACTGA-3’; SBE antisense 5’-TCAGTCTAGACATACT-3’ and Smad4 antibody. EMSA was performed by incubating 10 ug of nuclear extract in a 9 uL binding reaction mixture at 37 °C for 10 min. The binding reaction mixture for the super shift assay containing 1 uL of the non-diluted antibody of Smad4 was added to 1 uL of DIG-labeled double-stranded oligonucleotide and was incubated at 37 °C for 20 min, followed by the addition of 1 uL of the gel loading 10ul buffer at room temperature. The DNA-protein complexes were separated by electrophoresis in 6% non-denaturing polyacrylamide gels using 0.25 ul Tris-borate-EDTA as a running buffer. After electrophoresis, the gels were transferred to nylon membranes and detected chemiluminescent. The luminescent signals were analyzed using an ImageQuant LAS 4000 Scanner of GE Healthcare.

Mouse RPE protein lysates were prepared and analyzed by Western blotting as described previously (Yang et al, 2018 (link)). Briefly, mouse eyes were dissected to obtain the eyecup containing the RPE, choroid, and sclera. The eyecup was cut into four petals, incubated in RIPA lysis buffer containing protease inhibitors on ice for 45 min with occasional gentle tapping, and the insoluble fraction was removed by centrifugation. The supernatant was collected, the protein concentration was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific), and 20 μg of proteins were used for SDS–PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked in 5% nonfat dry milk in TBS with 0.1% Tween 20 (TBST) at room temperature for 30 min, incubated with a primary antibody (Table S2) in fresh 5% nonfat dry milk in TBST at 4°C overnight, washed in TBST, and incubated with an appropriate secondary antibody conjugated with HRP. The signals were detected with an ECL detection kit (RPN2232; GE Healthcare) using an ImageQuant LAS 4000 scanner (GE Healthcare). The intensity of each band was quantified using the ImageJ software (1.49v; NIH). The signal intensity of each protein was normalized by that of β-actin (protein/β-actin), and relative protein levels were calculated as the ratio of protein/β-actin of samples to that of control.

Ten to twenty micrograms of protein per condition were separated on a NuPage 4–12% Bis–Tris gel (WG1401, Thermo Fischer Scientific, Waltham, Massachusetts, USA). Proteins were transferred to nitrocellulose membrane by wet blotting. Membranes were blocked in 5% milk in TBS/0.1% Tween-20 for 1 h at room temperature and incubated with the primary antibody overnight at 4 °C. Primary antibodies were: mouse monoclonal anti-vinculin (Sigma, V9131) and anti-PP2A-C (in-house, gift from Dr. S. Dilworth, London, UK); rabbit anti-Snail (Cell Signaling Technologies, 3879T) and anti-Epcam (Abcam, AB71916). After washing in TBS/0.1% Tween-20, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (Dako for anti-mouse and Cell Signaling for anti-rabbit) for 1 h and developed using Western Bright ECL (Advansta) on the ImageQuant LAS4000 scanner (GE Healthcare). Primary antibodies were used in a dilution of 1/1000, the secondary antibodies were used in a dilution of 1/5000. All densitometric quantifications were done with Image Studio Lite software (version 5.2).