Thirty micrograms of total protein were loaded on 10% SDS-PAGE gels, transferred onto PVDF membrane and probed with primary antibodies against DDK tag (Origene, Rockville, MD, USA), ZO-1, occludin, and VE-cadherin (Invitrogen, Rockford, IL, USA), β1-Na+, K+-ATPase (Millipore, ...
PVDF membranes were blocked by incubating overnight at 4°C in 20 mls of SuperBlock blocking buffer (Pierce Biotechnology, Rockford, IL) in Tris-buffered saline (pH 7.4) containing 0.5% Tween-20 (TBST) with gentle shaking. Membranes were then washed three times for five minutes each with 20...
Cancer-associated fibroblasts (CAFs) are one of the most dominant components in the tumor stroma, which build up and remodel the extracellular matrix (ECM) structure [6]. As one of the major constituents of the tumor microenvironment (TME), CAFs play multiple roles in regulating tumorigenesis, ...
Seoul, Korea). Polyclonal rabbit anti Ets-1 and polyclonal mouse anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were horseradish peroxidase conjugated anti-mouse IgG and anti-rabbit IgG (Thermo Scientific Pierce, Rockford, IL, USA). ...
Mean cortical density of paired helical filament tau (PHFtau) was assessed with immunohistochemistry using an anti-phosphotau antibody (AT8, targeting Ser202/ Thr205 [Thermo Fisher Scientific, Rockford, IL, USA; 1:2000 dilution]) in the same eight regions to derive a quantitative PHFtau burden...
Liu XW, Taube ME, Jung KK, Dong Z, Lee YJ, Roshy S, Sloane BF, Fridman R, Kim HRC (2005) Tissue inhibitor of metalloproteinase-1 protects human breast epithelial cells from extrinsic cell death: a potential oncogenic activity of tissue inhibitor of metalloproteinases-1. Cancer Res 65(3):...
After adding the Halt protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford, IL), the cells were scrapped and collected. The chromatin preparation was then processed according to the manufacturer's protocol (Pierce Biotechnology). Lysis buffer was added and micrococcal nuclease ...
The amount of total protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of total protein (20 μg) was loaded and separated by SDS-PAGE. The protein was transferred to a nitrocellulose membrane, blocked and probed with appropriate antibodies. ...
(listed in Table3). After the membranes were incubated with HRP-labeled secondary antibodies at room temperature for 1 hour, the band images were developed with enhanced chemiluminescence (ECL) reagents (Pierce, Thermo Scientific, Rockford, IL, USA) and digitized on Image Quant LAS 4000 mini ...
The protein concentrations were detected using a BCA protein assay kit (Thermo Scientific, Rockford, IL). Protein digestion was performed using the filter-aided proteome preparation (FASP) method. After digested with sequencing grade trypsin (Promega, Madison, WI) at 37 °C overnight, the ...