Chemiluminescent hrp substrate kit

Cell lysates from DU145 and PC3 cell lines were prepared using RIPA buffer in the presence of protease inhibitor cocktails and Phosphatase Inhibitor Cocktails 2 and 3 (Sigma-Aldrich) as described previously29 (link). Proteins (40 μg) were loaded onto 5–12% Tris-acrylamide gels and blotted with the following antibodies: anti-STIM1 and ORAI1 (ProSci Inc.), anti-N-cadherin, E-cadherin and Apoptosis I Sampler Kit (BD Biosciences), anti-β-catenin and Wnt-1 (Abcam Inc.), anti-β-actin (Santa Cruz Biotechnology Inc.), anti-cyclin D1, DcR2, and Vimentin (Proteintech Group Inc.), anti-p-Smad3 (Epitomics Inc.), anti-claudin-1, Snail, p-Smad2 and Cell Cycle Regulation Antibody Sampler Kit II (Cell Signaling Technology Inc.), and horseradish peroxidase-conjugated secondary antibodies. The results were visualized using a chemiluminescent HRP substrate kit (Millipore) and analyzed using Image J software (National Institutes of Health, Baltimore, MD).The densitometry results were first normalized with that of β-actin and then compared with the control to obtain relative fold changes. The mean value for each blot was averaged from three independent experiments and indicated at the bottom of the blot.

Lung tissues were homogenized with lysis buffer consisting of 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 1 mM PMSF, and leupeptin 100 mg/mL. Homogenates were centrifuged, and the supernatant was taken. The protein concentrations were checked by the Bradford assay (Bio-Rad, Hercules, CA, USA). Ten to 15 μg of total proteins was loaded on 7.5–15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween-20. The membranes were incubated overnight at 4 °C with primary antibodies for β-actin (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), Mitofusin 1(1:300 dilution; Mfn1; sc-166644; Santa Cruz Biotechnology, Paso Robles, CA, USA), Mitofusin2 (1:300 dilution; Mfn2; #9482; Cell Signaling, Danvers, MA, USA), or Fis1 (1:300 dilution; sc-376447; Santa Cruz Biotechnology) and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Finally, blots were visualized with a chemiluminescent HRP substrate kit (Millipore, Billerica, MA, USA) via ChemiDoc (Bio-Rad). A β-actin (Bethyl Laboratories) antibody was used as an internal control. The intensity of the bands was determined by the ImageJ software (National Institutes of Health).

Equal amounts of proteins (20 μg) were mixed with 5× sample buffer and separated at RT under reducing conditions on 10% SDS-PAGE gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) for 1 h at RT and blocked with a solution containing 5% non-fat dry milk for 1 h at RT. Membranes were incubated overnight at 4 °C with primary antibody monoclonal mouse anti β-DG (1:500, ab49515, Abcam, UK) in 5% BSA. On the next day, membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:10,000, A3862, Sigma, USA) for 1 h at RT. Protein bands were visualized by exposing the membranes to a CCD camera (LAS1000, Fujifilm, USA) using a chemiluminescent HRP substrate kit (Millipore, USA). Membranes were stripped and re-probed with an anti-β-actin HRP-conjugate (1:75,000, A3854, Sigma, USA). After densitometric analysis using the ImageJ software (National Institute of Health, USA), primary antibody levels were normalized to β-actin expression.

Cells and tumor tissues were lysed in RIPA buffer containing protease inhibitors (Solarbio, Beijing, China). Proteins were size-fractionated on 10–12% PAGE gels and transferred to Immobilon-transfer nitrocellulose membranes (Millipore, Burlington, MA, USA). The membranes were incubated with anti-human NY-ESO-1 (1:250 dilution; Clone SP349; Abcam, Cambridge, MA, USA), anti-DNMT3 (1:2000; Clone EPR18455, Abcam), and anti-β-actin (1:2000 dilution; Clone 13E5; Cell Signaling Technology, Danvers, MA, USA), washed, and incubated with an HRP-labeled secondary antibody. Protein bands were detected with the Chemiluminescent HRP Substrate Kit (Millipore). The densitometry readings/intensity ratios were analyzed with the ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA) by comparing the protein band intensity of NY-ESO-1 or DNMT3a to the protein band intensity of β-actin.

Protein extracts from colon tissues were prepared by homogenization. Homogenates were centrifuged, and protein concentrations of supernatants were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Western blot was performed as previously described [15 (link)]. The primary antibodies included mouse anti-occludin (ThermoScientific), rabbit anti-zonula occludens (ZO)-1 (ThermoScientific), rabbit anti-toll-like receptor 4 (TLR4) (ThermoScientific), and rabbit anti-heat shock protein (HSP) 70 (Cell Signaling, Beverly, MA, USA). The membrane was subsequently incubated with horseradish peroxidase (HRP) conjugated-secondary antibodies. Finally, blots were developed with a chemiluminescent HRP substrate kit (Millipore, Billerica, MA, USA). The intensity of the bands was determined by Image J version 1.8.0 software (National Institutes of Health, Bethesda, MD, USA) and normalized with β-actin (Bethyl Laboratories, Montgomery, TX, USA) densitometric values.