After culture for 4 days, cells were lysed in RIPA lysis buffer (Solarbio, Beijing, China) and 1 mM PMSF (Solarbio, Beijing, China) on ice. The concentration of protein was metered using the BCA Protein Assay Kit (Thermo Scientific, MA, USA) and heat denaturation of protein in protein sample loading buffer (EpiZyme, Shanghai, China) at 100 °C for 10 min. Equal quantities of protein were separated on 10% SDS-PAGE gels by electrophoresis, transferred onto 0.45 um polyvinylidene-fluoride membranes (PVDF) (Immobilon, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in TBST buffer pH 7.5 for 1 h at room temperature, incubated with the first antibody. Anti-GAPDH, PI3K p85, PI3K p110, AKT, phosphorylated-PI3K p85 and phosphorylated-AKT were used as primary antibodies. All antibodies are listed in Additional file 6 : Table S1. Membranes were washed with TBST 3 times, 10 min each time, and incubated with corresponding secondary antibodies at room temperature for 1 h. Protein bands were visualized by ECL reagents (TransGen, Beijing, China).
Protein sample loading buffer
Manufactured by Ipsen
Sourced in China
Protein sample loading buffer is a solution used to prepare protein samples for analysis by gel electrophoresis. It facilitates the loading of protein samples into the sample wells of a gel and ensures uniform migration of proteins during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sds page, by Ipsen (2 mentions)
Ecl reagents, by Transgene (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Gapdh, by Transgene (1 mentions)
β tubulin, by Transgene (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Transgene (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
α sma, by Wuhan Servicebio Technology (1 mentions)
Collagen 1, by Abcam (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Irdye 680lt secondary antibody, by LI COR (1 mentions)
Biorad imager system, by Bio-Rad (1 mentions)
Western ecl substrate, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitors, by MedChemExpress (1 mentions)
Bca protein kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using protein sample loading buffer
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of Fibrosis Markers
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Tissue specimens and cells were lysed using RIPA buffer containing a protease inhibitors cocktail (Epizyme, Shanghai, China). Lysates were heated in 95 °C for 10 min in protein sample loading buffer (Epizyme). Total cell lysates were separated using 10% SDS-PAGE (Epizyme) and transferred to an Immobilon-P Transfer Membrane (Millipore, USA). Membranes were blocked using 5% nonfat milk dissolved in Tris-buffered saline and then incubated with a FN1 (CST, #26836, USA), ERK1/2 (CST, #4695, USA), p-ERK1/2 (CST, #4370, USA), TGF-β (CST, #3711, USA), COL3A1 (CST, #30565, USA), collagen I (Abcam, ab34170, USA) and α-SMA (Servicebio, GB11044, China) antibodies. The following day, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Transgen). GAPDH (Transgen) and β-tubulin (Transgen) were used as loading controls. Finally, membranes were visualized using an ECL western blotting detection reagent (Epizyme).
3
Western Blot Protein Analysis Protocol
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The cells and brain tissues were lysed with lysis buffer and incubated on ice for 20 min, and then homogenates were centrifuged (4 °C, 12,000 rpm, 20 min) to collect the supernatant. Samples (30 μg for total protein and 15 μg for membrane or synaptic fractionation) were boiled with the protein sample loading buffer (Epizyme, Shanghai, China) at 95 °C for 10 min. The samples were separated on 12.5% tris-glycine SDS-PAGE or 16% tris-tricine SDS-PAGE gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). After blocking with 5% nonfat milk, the membranes were rinsed with TBS and 0.1% Tween (TBST) for 5 min. Then, the membranes were incubated in primary antibody overnight at 4 °C. The membranes were washed with TBST 3 times, 5 min each time, and then were incubated in secondary antibody for 1.5 h at room temperature. The membranes were washed with TBST 3 times again, 5 min each. After removing the TBST, the blot was developed with Western ECL Substrate (Bio-Rad) and imaged with BIO-RAD Imager System (USA). The gray values of Western bands were analyzed quantitatively by Quantity One software. An Odyssey Imaging System (Licor, USA) was introduced to quantify fluorescence intensity when IRDye 800C secondary antibody (1:5000, 926-32211, Licor, USA) and IRDye 680LT secondary antibody (1:5000, 925-68020, Licor, USA) were used.
4
Protein Expression Analysis in Frozen Lung Tissues
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Frozen lung tissues were homogenized in RIPA lysis buffer (Beyotime, China). Then, the samples were placed on ice for 10 min and centrifuged for 15 min at 12,000 r/min. After adding 5× protein sample loading buffers (Epizyme, China), the supernatants of the samples were boiled for 10 min. Protein concentrations were detected with a BCA Protein Kit (Beyotime, China). The proteins were separated using 10% SDS-PAGE (Epizyme, China), then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) at a constant current of 200 mA. Next, after blocking with 5% non-fat milk for one hour, the membranes were incubated with different primary antibodies against p-AKT, AKT, GSK-3β, p-GSK-3β, TGF-β1, SNAIL1, E-cadherin, vimentin, TWIST1, fibronectin, N-cadherin, a-SMA, collagen I, and GAPDH at 4°C overnight. Subsequently, the membranes were incubated with the appropriate secondary antibodies. The blot was analyzed using the Image J software.
5
Protein Extraction and Western Blot Analysis
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Lung tissues or cells were fully lysed in RIPA buffer (Beyotime, China) containing a fresh mixture of protease and phosphatase inhibitors (MedChemExpress, United States).
The entire process was performed at 4°C. After centrifugation at 12000 r/min for 20 min, the supernatant was added to 5× protein sample loading buffers (Epizyme, China) and boiled for 10 min. A BCA protein kit (Thermo, USA) was used to determine protein concentrations. Denatured proteins were separated by 10% SDS-PAGE (Epizyme, China) and then transferred onto methanol-activated PVDF membranes (Millipore, USA) at a constant current of 400 mA. After blocking with 5% skim milk for 1 h, membranes were incubated with different primary antibodies overnight at 4°C. After washing the PVDF membrane several times, it was incubated with the appropriate secondary antibody (1:2,000) for 1 h at room temperature. Subsequently, ECL (GE Healthcare, United Kingdom) was used to visualize protein expression, and ImageJ software was used to analyze the band intensities.
The entire process was performed at 4°C. After centrifugation at 12000 r/min for 20 min, the supernatant was added to 5× protein sample loading buffers (Epizyme, China) and boiled for 10 min. A BCA protein kit (Thermo, USA) was used to determine protein concentrations. Denatured proteins were separated by 10% SDS-PAGE (Epizyme, China) and then transferred onto methanol-activated PVDF membranes (Millipore, USA) at a constant current of 400 mA. After blocking with 5% skim milk for 1 h, membranes were incubated with different primary antibodies overnight at 4°C. After washing the PVDF membrane several times, it was incubated with the appropriate secondary antibody (1:2,000) for 1 h at room temperature. Subsequently, ECL (GE Healthcare, United Kingdom) was used to visualize protein expression, and ImageJ software was used to analyze the band intensities.
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