Total protein was isolated from cells or liver samples using RIPA lysis buffer (50.0 mM Tris-HCl (pH = 8.0), 150.0 mM NaCl, 0.02% NaN3, 0.1% SDS, 1% NP-40, 0.5% deoxysodium cholate, 1 mM EDTA, 1 μM RIPA). The protein concentration was quantified with BCA kit (Pierce, ThermoFisher Scientific, USA). Thirty micrograms protein were separated by 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis depending on the band of target protein, then transferred onto equilibrated polyvinylidene difluoride membrane (PVDF, Immobilon®-PSQ transfer membranes, Merck Millipore, Germany). Membranes were incubated with primary antibodies overnight at 4 °C. Specific antibodies were listed in Supplementary Table 3 . After incubation with the secondary antibody, proteins were detected by enhanced chemiluminescence (GlarityTM Western ECL Substrate, Bio-Rad, USA) and the signals were recorded and quantified with Tanon-Image Software (Shanghai, China). Reference antibody GAPDH were used for data normalization.
Immobilon psq transfer membrane
Manufactured by Merck Group
Sourced in United States, Germany
Immobilon-PSQ transfer membrane is a laboratory equipment used for protein transfer and immobilization. It is a polyvinylidene fluoride (PVDF) membrane designed for high-performance protein transfer and immobilization in blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Beyoecl plus kit, by Beyotime (5 mentions)
Glarity western ecl substrate, by Bio-Rad (3 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
β actin, by Merck Group (2 mentions)
Ripa lysis solution, by Beyotime (2 mentions)
Hrp conjugated goat anti rabbit, by Beyotime (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Aprotinin, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate kit, by Merck Group (1 mentions)
Horseradish peroxidase hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Tgf β1, by Abcam (1 mentions)
Chemiluminescence reagent plus, by PerkinElmer (1 mentions)
26 protocols using immobilon psq transfer membrane
1
Western Blot Protein Quantification
2
Protein Extraction and Western Blot Analysis
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Total protein extracts from mTEC cells, GMC cells or kidney tissues using RIPA (Beyotime Biotechnology, China). The concentration of protein was assessed by the BCA Kit (Pierce, ThermoFisher Scientific, USA). Thirty micrograms of protein were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis depending on the band of the target protein. Next, the proteins in the gel were transferred onto an equilibrated polyvinylidene difluoride membrane (PVDF, Immobilon®-PSQ transfer membranes, Merck Millipore, Germany). Membranes were blocked by 5% skim milk and then interacted overnight with primary antibodies at 4 °C, following HRP-labeled secondary antibodies at room temperature for 1 h. The blots were detected by enhanced chemiluminescence (Glarity™ Western ECL Substrate, Bio-Rad, USA) and the signals were recorded and quantified via Tanon-Image Software (Shanghai, China). Data normalization was performed using reference antibody GAPDH or β-actin. The antibodies used for Western blotting are described in Supporting Information Table S4 .
3
Protein Extraction and Western Blot Analysis
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Total protein extracts from mTEC cells, GMC cells or kidney tissues using RIPA (Beyotime Biotechnology, China). The concentration of protein was assessed by the BCA Kit (Pierce, ThermoFisher Scientific, USA). Thirty micrograms of protein were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis depending on the band of the target protein. Next, the proteins in the gel were transferred onto an equilibrated polyvinylidene difluoride membrane (PVDF, Immobilon®-PSQ transfer membranes, Merck Millipore, Germany). Membranes were blocked by 5% skim milk and then interacted overnight with primary antibodies at 4 °C, following HRP-labeled secondary antibodies at room temperature for 1 h. The blots were detected by enhanced chemiluminescence (Glarity™ Western ECL Substrate, Bio-Rad, USA) and the signals were recorded and quantified via Tanon-Image Software (Shanghai, China). Data normalization was performed using reference antibody GAPDH or β-actin. The antibodies used for Western blotting are described in Supporting Information Table S4 .
4
Western Blot Protocol for Protein Analysis
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For all Western blots, the cell lysates were prepared in 1× SDS loading buffer. Total protein was applied to SDS-PAGE and transferred to Immobilon-PSQ Transfer Membranes (Merck Millipore). The membranes were blocked in 5% nonfat milk for 1 hour at room temperature, followed by the addition of specific primary antibodies overnight at 4°C. Horseradish peroxidase (HRP)–linked anti-rabbit IgG (catalog no. 7074P2, lot no. 26, Cell Signaling Technology) or HRP-linked anti-mouse IgG (catalog no. 7076P2, lot no. 32, Cell Signaling Technology) was used as a secondary antibody, corresponding to different proteins. Protein bands were detected using an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore).
5
Western Blot Analysis of Cellular Proteins
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Samples were loaded on 10% SDS-polyacrylamide gels for Western blot analysis and then transferred to Immobilon PSQ transfer membranes (Millipore, Bedford, MA, USA). Membranes were immediately incubated in TBS-T (Tris-HCl-based buffer containing 0.2% Tween 20, pH 7.5) containing 5% nonfat milk for 30 min at room temperature, washed, and incubated with primary antibodies: GFAP (mouse; 1:1000; Abcam, Cambridge, MA, USA), phospho-extracellular signal-regulated kinase (p-ERK) (rabbit; 1:1000, Cell Signaling Technology, Danvers, MA, USA), ERK (rabbit; 1:1000 Cell Signaling, MA, USA), p-p65 (rabbit monoclonal; 1:500, Cell Signaling MA, USA), and β-actin (mouse monoclonal; 1:10,000; Sigma-Aldrich) in TBS-T overnight at 4 °C. The membranes were then washed for 10 min and incubated with secondary monoclonal anti-mouse and anti-rabbit antibody conjugated with horseradish peroxidase (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS-T buffer for 2 h at room temperature. Membranes were developed by enhanced ECL and photographed using a cooled CCD camera system (ATTO Ez-Capture; Atto Corp., Tokyo, Japan). Fold changes in relative protein levels were quantified by densitometry using either total MAPK (ERK) or β-actin as loading controls.
6
Quantitative Western Blot Analysis
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Protein concentrations were determined using bicinchoninic acid (BCA) assay kit (Thermo Scientific, MA, USA) with bovine serum albumin standard. Samples (30 μg protein per lane) were separated in SDS-polyacrylamide gels and transferred electrophoretically to Immobilon-PSQ transfer membranes (Millipore), as reported previously57 (link). Band intensities were measured using FluorChemTM SP software (Alpha. Innotech, San Leandro, CA, USA) and normalized to β-actin or total Akt.
7
Western Blot Analysis of Ovarian Proteins
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Proteins were extracts from ovaries using Cell Lysis Buffer (Beyotime, P0013) for western blot analysis according to standard methods [56 (link)]. The proteins were separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membrane (PVDF, immobilon-PSQ transfer membranes, Millipore, ISEQ00010, USA). The membrane was blocked with TBST buffer (tris buffered saline, with tween-20) containing 5 - 10% BSA and incubated in primary antibody (Table S1 ) overnight at 4 °C. After three washes in TBST, the membrane was incubated with HRP conjugated goat anti-rabbit or anti-mouse IgG (Beyotime, A0216) diluted in TBST at room temperature for 1.5 hours. Finally, the membranes were reacted with BeyoECL Plus Kit (Beyotime, P0018). IPWIN software was used for density measurements.
8
Myoblast Differentiation and Emerin Expression
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Myoblasts were fixed with methanol (−20 °C). As a marker for proliferation Ki-67 (Thermo Scientific, RM-9106-S0), antibody was used [17] (link). Relocalization from the centrosome to the nuclear envelope of PCM1 has been found to be an early and reliable marker of differentiation [18] (link) and so was used to assess myoblast differentiation. For emerin immunofluorescence staining of myoblasts and biopsy sections MANEM1 antibody was used which recognizes emerin amino acids 89–96 (GYNDDYYE) [19] (link). For stainings with leucocyte type-specific antibodies the same fluorescently conjugated antibody set that was used for flow cytometry was employed; however, because the laser lines of the flow cytometer did not match the filter sets of the microscope, secondary antibodies were also used. All secondary antibodies were Alexafluor conjugated and generated in donkey with minimal species cross-reactivity. DNA was visualized with DAPI (4,6-diamidino-2 phenylindole, dihydrochloride). To determine if the small fragment encoded by the mutant allele is stably expressed, Western blot analysis optimized for small MW proteins was performed using Millipore Immobilon® PSQ Transfer Membranes and following the manufacturer's instructions. The blot was probed with monoclonal antibody MANEM14 against emerin amino acids 7–14 (LSDTELTT) [19] (link).
9
Western Blot Protein Analysis of Ovaries
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For each sample, proteins were extracted from 3 to 4 ovaries using the RIPA lysis solution (Beyotime, P0013C) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. They were then transferred onto a polyvinylidene fluoride membrane (immobilon-PSQ transfer membranes, Millipore, ISEQ00010, USA). After blocking in TBST buffer (TBS with Tween-20) containing 5% bovine serum albumin, the membrane was incubated in primary antibody (Table S2 ) overnight at 4 °C, washed and incubated with HRP-conjugated goat anti-rabbit (Beyotime, A0208) or anti-mouse IgG (Beyotime, A0216) diluted in TBST at room temperature for 1.5 h. Ultimately, the BeyoECL Plus Kit (Beyotime, A0018) was used for chemiluminescence; β-ACTIN was used as housekeeping protein as previous reported21 (link).
10
Western Blot Analysis of Protein Samples
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After washing, the collected pPGCLCs were lysed in RIPA lysis solution (Beyotime, P00113B) for 20 min on ice with vortexing. The extracted proteins were denatured by immersing in boiling water for 5 min and collected by centrifugation at 12 000 rpm for 3 min. The proteins were then separated by sodium dodecyl sulphate‐PAGE (SDS‐PAGE) and the proteins were electrophoretically transferred to poly‐vinylidene fluoride (PVDF) membranes (immobilon‐PSQ transfer membranes, Millipore, ISEQ00010, USA) with 200 mA for 150 min. After being blocked with 5% bovine serum albumin (BSA, Solarbio, A8020), the membranes were blocked with corresponding primary antibodies (Table S1 ) overnight. Following washes with TBST, membranes were incubated with secondary antibodies for 2 h (Table S1 ). Finally, the protein signalling was measured using a BeyoECL plus kit (Beyotime, P0018). GAPDH was selected as the housekeeping protein, and the data were analysed by AlphaView Sa Software (ProteinSimple, San Jose, CA, USA). The greyscales of the target proteins in different samples were analysed using the same area, and the greyscales of the target proteins were divided by that of the greyscales of the internal parameters to correct the errors. All the results were analysed without overexposure.
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