Total cellular proteins were extracted using RIPA cell lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors and phosphatase inhibitors (Bimake, Shanghai, China), and protein concentrations were quantified by a BCA protein assay kit (Beyotime, Shanghai, China). Then, Western blot analysis was performed as described previously,19 (link) and a total of 30 µg of protein from each sample was used to detect proteins of interest. The primary antibodies for Western blotting were as follows: anti-ACVR2A (Abcam, Cambridge, UK), anti-Snail (CST, Danvers, MA, USA), anti-Slug (CST, Danvers, MA, USA), anti-Vimentin (CST, Danvers, MA, USA), anti-Smad2/3 (CST, Danvers, MA, USA), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (CST, Danvers, MA, USA), anti-E-cadherin (BD Biosciences, NJ, USA), anti-Flag (MBL, Japan), anti-GAPDH (Proteintech, Wuhan, China) and anti-β-actin (Proteintech, Wuhan, China).
Protease inhibitors and phosphatase inhibitors
Manufactured by Selleck Chemicals
Sourced in United States, China
Protease inhibitors and phosphatase inhibitors are lab equipment used to inhibit the activity of proteases and phosphatases, respectively. Protease inhibitors prevent the breakdown of proteins by proteolytic enzymes, while phosphatase inhibitors block the dephosphorylation of molecules by phosphatases. These products are commonly used in research applications to maintain the integrity of proteins and protein modifications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Anti gapdh, by Proteintech (2 mentions)
Enhanced chemiluminescent substrate kit, by Yeasen (2 mentions)
Bca protein assay reagent, by Yeasen (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti acvr2a, by Abcam (1 mentions)
Ripa cell lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti e cadherin, by BD (1 mentions)
Bca reagent, by Beyotime (1 mentions)
Ripi lysis buffer, by Beyotime (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Du800 spectrophotometer, by Beckman Coulter (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Histone h4, by Abcam (1 mentions)
Azgp1, by Abcam (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
7 protocols using protease inhibitors and phosphatase inhibitors
1
Western Blotting of EMT Markers
2
Piezo1 Protein Expression Analysis
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Cells were lysed in RIPI Lysis Buffer (Beyotime) supplemented with protease inhibitors and phosphatase inhibitors (Bimake). Protein concentrations were measured with the BCA reagent (Beyotime) by using a Beckman Coulter DU‐800 spectrophotometer. Equal amounts of protein were resolved by SDS‐PAGE and immunoblotted with Piezo1 Monoclonal Antibody (Invitrogen, MA5‐32876) at a dilution of 1:1000. The immunoblots were detected using an Odyssey Fc Imaging System (LI‐COR).
3
Western Blot Analysis of Protein Lysates
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Cells were lysed for 30 min in ice with lysis buffer (Beyotime Biotechnology) containing protease inhibitors and phosphatase inhibitors (Bimake). After centrifugation at 16,000×g for 15 min at 4 °C, the protein concentrations were determined by the BCA method (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). Add the proteins with 5 × loading buffer and use 1 × loading buffer to make sure all the samples the same concentration. Heating samples at 100 °C for 10 min for denaturation. The samples were resolved by SDS/PAGE, transferred to PVDF membranes (Millipore, Massachusetts, USA). The membranes were blocked with 5% (w/v) skimmed milk in TBS at room temperature for 1 h, then probing with antibodies at 4 °C overnight. Using immune blotting to analysis by HRP-conjugated secondary antibodies. Information on the antibodies is provided in Additional file 1 : Table S3. A chemiluminescent (ECL) chromogenic substrate was used to visualize the bands (Solarbio). Visualization was performed using the hemic Doc™ XRS Imager (Bio-Rad, USA). The uncropped blots are shown in Additional file 1 : Fig. S9.
4
Western Blot Analysis of Protein Expression
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The CD24, SNAIL, SLUG, E-cadherin, PTEN, phosphorylated AKT, total AKT, ACTIN, and GAPDH primary antibodies were purchased from Cell Signaling Technology (1:1000, Beverly, MA, USA). The AZGP1, histone H4, and PRP primary antibodies were purchased from Abcam (1:1000, Cambridge, UK). Following standard procedures, we lysed cells using RIPA buffer (Beyotime, Nantong, China) with protease inhibitors and phosphatase inhibitors (Bimake, Houston, TX, USA). The protein concentrations of the cell lysates were quantified by using the BCA protein assay kit (Thermo Fisher, Waltham, MA, USA). Finally, 20 mg protein was separated by 10% SDS-PAGE and then transferred into polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% skim milk for 1 h at room temperature and then probed with the primary antibodies over night at 4 °C. Next, the membranes were washed with TBST and incubated with the secondary antibodies, which were conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. A luminescent image analyzer (ImageQuant LAS4000 mini) was utilized to detect the protein. All these experiments were performed in triplicate. Original blots can be found at supplementary File S1 .
5
Protein Expression Analysis in Liver
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Liver tissues were lysed in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) for total protein according to the manufacturer’s instructions (Shenergy Biocolor Bioscience & technology CO., Shanghai, China). Protein concentration was measured using BCA Protein Quantitative Assay Kit (Shenergy Biocolor Bioscience & technology CO., Shanghai, China). The target proteins were blotted with the following antibodies: anti-HNF4α (41770, Invitrogen, USA), anti-SIRT1 (sc-15404, Santa Cruz Biotechnolosy, USA), anti-PPARα (MAB3890, Merck KGaA, Germany), anti-GAPDH (60004–1, Proteintech, China). The relative target protein levels were quantified by densitometry normalized to GAPDH on the same membrane. The samples in one group from three different mice and three independent experiments were performed.
6
Cell Lysis and Protein Detection
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Modified RIPA buffer (50 mM Tris–HCl, pH7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) was utilized to lyse cells. Protein concentrations were detected by BCA protein assay reagent (Yeasen, Shanghai, China). Cellular extracts were resolved through SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, USA), then incubated with the indicated primary antibodies. Enhanced chemiluminescent substrate kit (Yeasen) was used to analyze corresponding antibody specific signals. Table S4 lists the antibodies used.
7
Protein Extraction and Co-Immunoprecipitation
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Modi ed RIPA buffer (50 mM Tris-HCl, pH7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) was utilized to lyse cells. Protein concentrations were detected by BCA protein assay reagent (Yeasen, Shanghai, China). Cellular extracts were resolved through SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, USA), then incubated with the indicated primary antibodies. Enhanced chemiluminescent substrate kit (Yeasen) was used to analyze corresponding antibody speci c signals. Table S5 lists the antibodies used.
Co-immunoprecipitation (Co-IP) 2 × 10 7 RBE and HEK293T cells washed by PBS then were harvested and lysed with NP40 lysis buffer (Solarbio, N8031, Beijing, China) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, Houston, USA). Then the lysates incubated with Flag-, EGLN3-antibody and control IgG after centrifugation respectively in a rotating incubator overnight at 4 °C. Subsequently, the cell lysates were incubated with Protein A/G (Sigma-Aldrich, St. Louis, MO, USA) for another 3 h. Afterwards, the Protein A/G Dynabeads were eluted and collected. The eluent was boiled and denatured for immunoblotting.
Co-immunoprecipitation (Co-IP) 2 × 10 7 RBE and HEK293T cells washed by PBS then were harvested and lysed with NP40 lysis buffer (Solarbio, N8031, Beijing, China) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, Houston, USA). Then the lysates incubated with Flag-, EGLN3-antibody and control IgG after centrifugation respectively in a rotating incubator overnight at 4 °C. Subsequently, the cell lysates were incubated with Protein A/G (Sigma-Aldrich, St. Louis, MO, USA) for another 3 h. Afterwards, the Protein A/G Dynabeads were eluted and collected. The eluent was boiled and denatured for immunoblotting.
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