At 72 h following transfection, the cells were harvested while the lysates of the cell were generated using RIPA lysis buffer for approximately 30 min at 4°C. The BCA Protein Assay kit (Pierce; Thermo Fisher Scientific) was used to determine the protein concentration. Subsequently, 12.5% SDS-PAGE was used to separate the total protein evenly (50 µg/lane) followed by transfer to PVDF membranes. Membranes were blocked with 5% skim milk powder in TBS-T for 2 h at room temperature and then incubated with primary antibodies at 4°C overnight. The primary antibodies were obtained from Abcam [anti-β-actin antibody (diluted at 1/5,000, ab6276), anti-CCND1 (diluted at 1/200, ab16663) and rabbit anti-CDCA7L (diluted at 1/2,000, ab70637)]. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000, ab6721; Abcam) at room temperature for 1 h. β-actin was used as an internal control for the normalization of the protein expression levels. The proteins bands were visualized using an Enhanced Chemiluminescence Plus Western Blotting Detection system (GE Healthcare). Band intensities were quantified by densitometry using ImageJ Software version 1.6 (National Institutes of Health).
Enhanced chemiluminescence plus western blotting detection system
Manufactured by GE Healthcare
Sourced in United States
The Enhanced Chemiluminescence Plus Western Blotting Detection system is a laboratory equipment product that detects and quantifies proteins in Western blot analysis. It utilizes a chemiluminescent reaction to generate light signals proportional to the amount of target proteins present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (3 mentions)
Las 4000epuv mini luminescent image analyzer, by GE Healthcare (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ab16663, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ab47267, by Abcam (1 mentions)
Alpha tubulin, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay buffer, by Fujifilm (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp linked anti rabbit igg antibody, by GE Healthcare (1 mentions)
Anti rat igg antibody, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Polyacrylamide gel, by ATTO Corporation (1 mentions)
Nuclear extraction kit, by Cayman Chemical (1 mentions)
Lysis buffer, by Yeasen (1 mentions)
Precast sds page gel, by Bio-Rad (1 mentions)
10 protocols using enhanced chemiluminescence plus western blotting detection system
1
Western Blot Analysis of Transfected Cells
2
Western Blot Analysis of Protein Samples
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Cells (5 × 106) were lysed for 20 min with lysis buffer (Beyotime Biotechnology) containing protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations were determined by the BCA method (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). After centrifugation at 16,400×g for 15 min at 4 °C, the samples were resolved by SDS/PAGE, transferred to PVDF membranes (Immobilon-P membrane, Millipore, Massachusetts, USA), and analysed by immune blotting using HRP-conjugated secondary antibodies. The membranes were blocked with 5% (wt/vol) skimmed milk in TBS plus Tween 20 at 4 °C overnight before probing with antibodies. Information on the antibodies are provided in Supplementary Table 3 . An enhanced chemiluminescent (ECL) chromogenic substrate was used to visualize the bands (Pierce). Visualization was performed using the Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare, Connecticut, USA) and LAS-4000EPUV mini Luminescent Image Analyzer (GE Healthcare). The uncropped blots are shown in Supplementary Fig. 14 .
3
Western Blot Analysis of CBX3 Protein
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The cells and tissues were harvested according to the manufacturer's instructions. A BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. Afterwards, equal amounts of total protein (50 µg/lane) were separated by 12.5% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% skim milk powder in TBS-Tween (0.1%) for 2 h at room temperature and then incubated with a 1:800 dilution of rabbit anti-CBX3 (cat. no. ab10480) antibody or a 1:2,500 dilution of rabbit anti-GAPDH (cat. no. ab9485) antibody (Abcam, Cambridge, MA, USA). Additionally, the secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; cat. no. ab6721; Abcam). The positive bands were detected using the Enhanced Chemiluminescence plus western blotting detection system (GE Healthcare, Chicago, IL, USA). Band intensities were quantified by densitometry using ImageJ Software version 1.6 (National Institutes of Health, Bethesda, MD, USA). GAPDH was applied as the internal reference for the normalization of the expression levels of CBX3.
4
Overexpression of SHISA2 in PC-3 Cells
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PC-3 cells (1×106) were seeded onto a 10-cm dish and cultured to 50% confluence. Cells were transfected with 10 µg of the pIRESneo3 empty vector or pIRESneo3-SHISA2-HA expression vector using FuGENE6 reagent (Roche Diagnostics), according to the manufacturer's protocol. Cells were lysed using IP Lysis Buffer (Thermo Fisher Scientific, Inc.) and protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell lysates (30 µ (30 es and culture medium (DMEM without FBS) were separated on SDS-PAGE (12% gels), transferred to polyvinylidene difluoride (PVDF) membranes, and subsequently blocked with 5% milk for 30 min at RT. Membranes were then incubated with anti-HA mAb or anti-SHISA2 pAb for 60 min at RT. Signals were visualized using the Enhanced Chemiluminescence Plus Western Blotting Detection system (GE Healthcare). The anti-ACTB antibody (dilution, 1:5,000; clone AC-15; Sigma-Aldrich; Merck KGaA) served as a loading control. ACTB was used as a loading control (cell lysate). Silver stain was used as a loading control (medium).
5
Western Blot Analysis of PDX1 Protein
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Cells were washed with cold PBS and lysed in radioimmunoprotein-assay (RipA) buffer (10 mM Tris/HCl, 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate and protease inhibitors, pH 7.4), and maintained under constant agitation for 30 min. Cell extracts were then centrifuged at 16,000× g for 20 min at 4 °C). Protein concentration was determined by the BCA protein assay kit according to the manufacturer’s instructions. Equal amounts of protein were resolved on 10% SDS-PAGE under reducing conditions and proteins were transferred to a nitrocellulose membrane. Blots were incubated with primary antibodies directed against PDX1 (1:1000, Abcam, ab47267, Cambridge, UK), alpha-tubulin (1:1000, Invitrogen, 32-2700, Waltham, MA, USA), washed three times with PBS-0.05% tween and followed by incubation with secondary antibodies, directed against goat anti-mouse or rabbit HRP-conjugated (1:5000, Sigma-Aldrich). The visualization of immunoreactive bands was performed using the enhanced chemiluminescence plus Western blotting detection system (GE Healthcare). Quantification of protein signal intensity was performed by volume densitometry using ImageJ 1.47t software (NIH, Bethesda, MD, USA).
6
Western Blot Analysis of Transcription Factor Expression
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Western blotting was performed to analyze TF expression in cultured cells. Whole-cell lysates were prepared using radioimmunoprecipitation assay buffer (Wako Pure Chemical Industries, Osaka, Japan) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Total protein concentration was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). Protein samples (35 μg) were separated on a 4%-20% polyacrylamide gel (ATTO Corporation, Tokyo, Japan) and transferred on to Immobilon-P membrane (Millipore, Billerica, MA, United States). As primary antibodies, anti-TF 1849 (generated by us) and a commercially available goat anti-human actin (C-11) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, United States), were used. A horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody and anti-rat IgG antibody (GE Healthcare, Little Chalfont, United Kingdom) were used as the secondary antibodies. Immunoreactive bands were visualized using the Enhanced Chemiluminescence Plus Western blotting detection system (GE Healthcare).
7
Western Blot Analysis Protocol
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Cells were rinsed with ice-cold PBS and lysed by radio immunoprecipitation assay lysis buffer with protease and phosphatase inhibitors for 20 minutes on ice. Then the cells were centrifuged at 12 000 × g for 10 minutes at 4°C. Protein extracts (20 µg) were resolved using SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 200 V, 45 minutes). The protein bands were electro-transferred to nitrocellulose membranes (80 V, 120 minutes). Membranes were then treated with a 5% enhanced chemiluminescence blocking agent (GE Healthcare Bio-Sciences, Piscataway, NJ) in saline buffer (T-Tris-buffered saline) containing 0.1% Tween-20, 10 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2 at a pH of 7.4 for 1 hour, and then incubated with the primary antibody overnight at 4°C. Subsequently, membranes were washed 3 times in T-Tris-buffered saline and bound antibodies were detected using appropriate horseradish peroxidase-conjugated secondary antibodies, followed by analysis in an enhanced chemiluminescence plus Western blotting detection system (GE Healthcare Bio-Science).
8
Assessing Autophagy and Oxidative Stress
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Protein expression was detected via western blotting. The total and nuclear proteins in harvested cells were extracted using a total protein extraction solution (iNtRON Biotechnology, Seoul, Korea) and a nuclear extraction kit (Cayman Chemical Company, Ann Arbor, MI, USA), respectively, following protein level quantification. Cell lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated proteins were transferred onto nitrocellulose membranes. The protein-coated membranes were blocked with 3% bovine serum albumin and incubated with primary antibodies against Atg5, beclin-1, Atg7, LC3, p62, HO-1, Cu/Zn SOD, actin, phospho-Nrf2, Nrf2, and TBP for 2 h. Membranes were washed thrice with 1×Tris-buffered saline and Tween 20 (TTBS), incubated with secondary antibodies for 1 h, and then washed with 1× TTBS. Protein bands were detected using the enhanced chemiluminescence plus western blotting detection system (GE Healthcare Life Sciences, Buckinghamshire, UK).
9
Western Blot Analysis of CAL-62 Cells
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We used lysis buffer (YEASEN, Shanghai, China) with protease inhibitors (Roche, Indianapolis, IN, USA) to lyse CAL-62 cells. Protein concentrations were determined by the BCA method (Pierce, Therrmo Fisher Scienti c Inc., Rockford, IL, USA). The protein samples were subjected to SDS/PAGE and transferred to PVDF membranes (Immobilon-P membrane, Millipore, Massachusetts, USA). The membranes were blocked with 5% skimmed milk in TBS plus Tween 20 at room temperature for 1 hour, followed by incubation with target antibodies at 4 °C overnight. Information on the antibodies are provided in Table S3. After incubation with HRP-conjugated secondary antibodies for 1 hour, visualization of the protein bands was achieved by an enhanced chemiluminescent chromogenic substrate using the Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare, Connecticut, USA) and LAS-4000EPUV mini Luminescent Image Analyzer (GE Healthcare).
10
Western Blot Protein Quantification
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Immunoblot was performed as described previously (Blanchet et al., 2012) . Briefly, cells were washed using cold PBS and lysis was performed by using 50 mM Tris-HCl (pH 8), 137 mM NaCl, 10% glycerol, 1% NP-40 and phosphatase, protease, and deacetylase inhibitors (Sigma-Aldrich) on ice. Western blotting was performed using 30 mg of proteins loaded on SDS-PAGE precast gel (Bio-Rad). After electrotransfer, the membrane was blocked for 1 hr at room temperature with 5% nonfat milk in 0.1% Tween Tris-buffered saline (TTBS) buffer. Membranes were then incubated overnight at 4 C with primary antibodies as indicated in blocking buffer containing 5% nonfat milk at the dilution specified by the manufacturers. Membranes were then incubated with the secondary antibody conjugated with the enzyme horseradish peroxidase. The visualization of immunoreactive bands was performed using the enhanced chemiluminescence plus western blotting detection system (GE Healthcare). Quantification of protein signal intensity was performed by volume densitometry using ImageJ 1.47t software (NIH).
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