The total protein was extracted from LX-2 cells prepared with ice-cold lysis buffer (Biosharp). The protein concentrations of the cell lysates were determined with a BCA protein kit (Beyotime Institute of Biotechnology, China). Samples containing equal amounts of protein were mixed with loading buffer containing 5% 2-mercaptoethanol and then heated for 10 min at 95 °C. Twenty to thirty micrograms of protein lysates was separated on 6–12% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (Millipore). TBST containing 5% nonfat milk was used to block nonspecific binding for 2 h at room temperature. Next, the membranes were incubated according to the instruction with the respective primary antibodies overnight at 4 °C and then treated with the appropriate HRP-conjugated secondary antibodies (1:5,000 dilutions). The signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (CLINX, Shanghai, USA), according to the manufacturer’s protocols. The experiments were repeated three times.
Lysis buffer
Manufactured by Biosharp
Sourced in China
Lysis buffer is a solution designed to disrupt cellular membranes and release the contents of cells, including proteins, nucleic acids, and other cellular components. It serves as a crucial component in various biological and biochemical processes, enabling the extraction and isolation of these cellular components for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bca protein kit, by Beyotime (2 mentions)
Sirt3, by Merck Group (2 mentions)
Ecl western blotting reagent, by Merck Group (2 mentions)
Non fat milk, by Merck Group (2 mentions)
Protease inhibitor cocktail, by MedChemExpress (2 mentions)
Non fat milk, by Bio-Rad (2 mentions)
Ecl solution, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
β actin, by Abcam (2 mentions)
Sirt2, by Merck Group (1 mentions)
Pepck1, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti mouse and anti rabbit antibodies, by Cell Signaling Technology (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
Mmp 9, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Ccd camera, by Clinx (1 mentions)
12 protocols using lysis buffer
1
Western Blot Analysis of Protein Extracts
2
Western Blot Analysis of Protein Expression
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Cultured cells were lysed with ice-cold lysis buffer (Biosharp). Extracted proteins were mixed with loading buffer containing 5% 2-mercaptoethanol and then denatured at 100°C for 10 minutes. Samples were separated on 8% to 12% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes (Millipore). Afterwards, membranes were incubated in TBST containing 5% nonfat milk for 2 hours at room temperature. Next, membranes were incubated according to instructions with the respective primary antibodies overnight at 4°C and then treated with the appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:3000 dilutions). The blots were developed with ECL Western blotting reagents (Millipore). HRP-conjugated anti-mouse and anti-rabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA). The following antibodies were used: SIRT1 (sc15404, Santa Cruz), SIRT2 (s8447, Sigma), SIRT3 (s4072, Sigma), β-Catenin (ab32572, Abcam), E-Cadherin (3195s, CST), PEPCK1 (sc32879, Santa Cruz), JNK (9258, CST), p-JNK (4668, CST), ERK (4695, CST), p-ERK (4370, CST), RAS (3965, CST), MMP-9 (ab137867, Abcam), actin (A5441, Sigma), Tubulin (2144, CST), Ac-Tubulin (5335, CST), anti-mouse IgG (7076, CST), and anti-rabbit IgG (7074, CST).
3
Western Blot Analysis of Protein Extracts
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The total protein were extracted from target tissues and cells prepared with ice-cold lysis buffer (Biosharp). The supernatant was used for Western blot analysis. Protein concentrations were determined using a BCA protein kit (Beyotime Institute of Biotechnology, China). Samples containing equal amounts of protein were mixed with loading buffer containing 5% 2-mercaptoethanol and then heated for 10 min at 95°C. Twenty to forty micrograms of protein lysates were separated on 8–12 % sodium dodecyl sulfate-polyacrylamide gels and then transferred to the NC (Nitrocellulose) membranes (Millipore, Bedford, MA, USA). Tris Buffered Saline, with Tween-20 (TBST) containing with 5% nonfat milk or bovine serum albumin was used to block nonspecific binding for 2h at room temperature. Then, the membranes were incubated with the primary antibodies. Subsequently, the membranes were rinsed three times with Tris Buffered Saline (TBS) and 0.1% Tween-20 (TBS-T) for 10min and re-incubated for 1h at room temperature in blocking buffer with each Horseradish Peroxidase (HRP)-conjugated secondary antibody (1:5000), then washed three times for 10min each. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (CLINX, Shanghai, China). Data are representative of at least three independent experiments.
4
Western Blot Analysis of Metabolic Proteins
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The cells were lysed using lysis buffer (Biosharp) to obtain total protein. Next, loading buffer containing 5% 2‐mercaptoethanol was mixed with the lysate. The proteins were denatured at 100°C for 10 minutes before being separated on 8%‐12% sodium dodecyl sulfate‐polyacrylamide gels. After transfer to PVDF membranes (Millipore), the membranes were incubated for 2 hours at room temperature in TBST containing 5% skim milk. Afterward, membranes were incubated with the corresponding primary antibodies overnight at 4°C according to the manufacturer's instructions. After treatment with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies, the membranes were incubated with ECL western blotting reagents (Millipore). The following antibodies were used: SIRT3 (s4072; Sigma), cMYC (ab32072; Abcam), HIF1α (36169; CST, Danvers, MA, USA), PDK1 (5662; CST), p‐PDHA1 (ab92696; Abcam), GAPDH (ab128915; Abcam), Actin (A5441; Sigma), PDHA1 (ab110334; Abcam), GLUT1 (ab115730; Abcam), Anti‐mouse IgG (7076; CST), and Anti‐rabbit IgG (7074; CST).
5
DROSHA Protein Expression Analysis
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Lysis buffer (Biosharp, Hefei, China) including 5 g/L sodium deoxycholate, 0.2 g/L NaN3,10 mL/l NP-40, 150 mmol/L NaCl 100 μg/mL, 0.1 g/LSDS, phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, and 50 mmol/L Tris-HCl (pH 8.5) was used to lyse the cells or tissues according to the manufacturer’s instructions. We used 12% SDS-PAGE to purify protein samples and then transferred them to nitrocellulose membranes (GE Healthcare, Milan, Italy). To avoid unspecific binding, Tris-buffered saline/Tween-20 (0.1%, Bioeasy, Shanghai, China) including 5% non-fat milk (Merck, Darmstadt, Germany) was used to incubate with the membrane at room temperature for 2 h. Subsequently, monoclonal antibodies against DROSHA (anti-DROSHA antibody, 1:1000, RT, 2h, Abcam, Boston, MA) were incubated with the blot, and at the same time a monoclonal antibody against β-actin (anti-β-actin antibody, 1:10000, RT, 1 h, Abcam, Boston, MA) were used as an internal control. After washing with PBS (Invitrogen, CA), secondary antibody conjugated to HRP (1:10000, RT, 1 h, Abcam, Boston, MA) and the ECL Western Blotting Kit (Promega, Milan, Italy) were used for signal detection according to the manufacturers’ protocols.
6
Western Blot Analysis of Protein Lysates
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Cells or tissues were lysed in lysis buffer (Biosharp, China) supplemented with a protease inhibitor cocktail (MedChemExpress, USA) and phosphatase inhibitor cocktail (MedChemExpress, USA) and clarified by centrifugation at 12,000 × g for 20 min. The protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific, USA). Equal amounts of the protein lysate were separated by 10% SDS‒PAGE gel, transferred onto PVDF membranes (Bio-Rad, USA), and incubated in 5% nonfat milk (Bio-Rad, USA) at room temperature. The membranes were incubated with primary antibodies at 4°C overnight and then hybridized with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals were visualized with ECL solution (Bio-Rad, USA) and quantified by analyzing the integrated density, which was normalized to the level of β-actin using ImageJ software (NIH, USA). The antibodies used are shown in Table S3 .
7
Quantitative Western Blot Analysis of SPAG5, Cyclin D1 in Liver Cancer Cells
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Protein extraction of Huh7 and HCCLM3 cell was accomplished using a lysis buffer (Biosharp Life Sciences), followed by quantification using the BCA method. Proteins (50 µg) were separated on 8% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 µm; Abiowell). The PVDF membranes were then blocked with 5% skim milk for 1.5 h at room temperature. Overnight incubation at 4°C on a shaker followed, with antibodies against SPAG5 (1:20,000; cat. no. 14726-1-AP; ProteinTech Group, Inc.), cyclin D1 (1:20,000; cat. no. ab134175; Abcam), and GAPDH (1:20,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). Post-0.05% TBST washing, the membranes underwent incubation with goat anti-rabbit IgG-HRP (1:20,000; cat. no. PR30011; ProteinTech Group, Inc.) at 37°C for 1 h. The membrane was visualized using an enhanced chemiluminescence (ECL) detection system (Biosharp Life Sciences). The band grayscale value is calculated using ImageJ (1.53a; National Institutes of Health).
8
Protein Expression Analysis Protocol
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Treated cells were lysed using a lysis buffer (Biosharp) and centrifuged (4°C, 12000 × g, 20 min). The supernatant was collected and the protein concentration was calculated using a protein quantification kit (Biosharp). The protein samples were subsequently boiled (95°C, 5 min) and then cooled down. A sample containing 60 µg of protein was added to each well for electrophoresis (80 V, 30 min; 120 V, 2 h). Subsequently, proteins were transferred onto a membrane (Millipore, MA, United States; 200 mA). The membranes were blocked in 5% non-fat milk (Biosharp) for 2.5 h, washed three times with TPBS (PBS contains 0.1% Tween 20), and incubated with primary antibodies (Ki67: Proteintech, #27309-1-AP, Wuhan, China; Bcl-2:CST, Boston, MA, United States, #15071T; Bax:CST, #5023T; p62:CST, #8025T; LC3-2:CST, #4108S; Beclin1:CST, #3495T; PI3K: CST, #4249T; AKT: CST, #4691T; mTOR: CST, #2983T; p-PI3K: CST, #17366S; p-AKT: CST, #4060S; p-mTOR: CST, #5536S) overnight at 4°C. The membranes were then washed and co-incubated with the corresponding secondary antibodies (Proteintech, #SA00001-1, SA00001-2, Wuhan, China) for 2 h at 20°C, and the protein bands were exposed using the ECL developing fluid (Millipore).
9
Protein Quantification and Western Blot
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We collected the cells and lysed them in lysis buffer (Biosharp, Hefei, Anhui, China) containing 5 g/l sodium deoxycholate, 10 ml/l NP-40, 1 μg/ml aprotinin, 100 μg/ml phenylmethylsulfonyl fluoride, 0.1 g/lSDS, 0.2 g/l NaN3, 150 mmol/l NaCl, and 50 mmol/lTris-HCl (pH 8.5). Cell lysates were clarified of cell debris by centrifugation at 20 800 × g for 15 min through a QIAshredder spin column assembly (Qiagen) at 4°C. The Bradford method was employed to determine the concentrations of protein. The proteins were loaded on a 12% polyacrylamide gel (SDS-PAGE) and electrophoresed. After blotting onto a polyvinylidene fluoride membranes (Millipore, Boston, Massachusetts), we blocked the proteins with 5% non-fat milk (Merck, Darmstadt, Darmstadt, Germany) in Tris-buffered saline/Tween-20 (0.1%, Bioeasy, Shanghai, China) at room temperature for 2 h. The membranes were then probed with primary antibodies against ET-1 (1:10,000; Z0334; Dako), and β-actin (1:10,000; Abcam, Cambridge, LON, UK). Immunoreactive bands were quantified individually by estimating the number of pixels using an image analysis system (Image J, NIH, MD). The band intensity of the target protein was first normalized with the band intensity of the β-actin and expressed as a ratio of the 2 bands.
10
Protein extraction and western blotting
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Tissues or cells were lysed in lysis buffer (Biosharp, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (MedChemExpress, USA) and clarified by centrifugation at 12,000 × g for 15 min. The protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific, USA). Protein samples (30 μg) were fractionated with SDS-PAGE, transferred to PVDF membranes (Bio-Rad, USA), and incubated in 5% nonfat milk (Bio-Rad, USA) at room temperature. The membranes were incubated with primary antibodies at 4 °C overnight and were then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals were visualized with ECL solution (Bio-Rad, USA) and quantified by analysis of the integrated density normalized to the level of β-actin using ImageJ. The antibodies used are shown in Supplementary Table S2 .
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