Total protein extraction was performed as previously described [18 (link)]. The protein concentration was determined by the BCA method. Proteins were separated by SDS-PAGE and then transferred to PVDF membranes (0.45 mm, Millipore). The PVDF membranes were washed with TBST, blocked for 1 h with 5% skim milk powder dissolved in TBST and incubated with primary antibodies against TGF-β (ab170874, Abcam), TNF-α (ab1793, Abcam), p-NFKB (ab86299, Abcam), and oxLDL (ab14519, Abcam) at 4°C overnight with dilutions recommended by the manufacturer. GAPDH was used as an internal reference. The PVDF membranes were washed with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at 37°C for 1 h. Protein bands were detected using chemiluminescence (ECL) reagent (Pierce, Thermo Scientific). The quantitative analysis of protein band density was performed on Quantity One (Bio-Rad).
Pvdf membranes 0.45 mm
Manufactured by Merck Group
Sourced in Germany, United Kingdom
PVDF membranes (0.45 mm) are a type of laboratory filter membrane made from polyvinylidene fluoride (PVDF). They have a pore size of 0.45 micrometers and are commonly used for filtration and separation applications in research and scientific settings.
Automatically generated - may contain errors
Lab products found in correlation
Ab14519, by Abcam (1 mentions)
Ab170874, by Abcam (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Ab1793, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
Cell counting kit 8 cck 8, by Transgene (1 mentions)
Western blot luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab11370, by Abcam (1 mentions)
Ab47003, by Abcam (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
4 protocols using pvdf membranes 0.45 mm
1
Quantitative Protein Expression Analysis
2
Western Blot and Cell Proliferation Assay for INS-1 β Cells
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INS-1 β cells were harvested and lysed to perform western blot routinely. 80μg of total protein were electrophoresed on a 10% (w/v) SDS-PAGE and transferred into PVDF membranes (0.45 mm, Millipore, Darmstadt, Germany). Antibodies used were as follows: rabbit anti-IRS-1, anti-IRS-2 (both from Immuno Way; both diluted 1:200); rabbit anti-GAPDH (ZhongShan, Beijing, China, 1:1000); HRP-conjugated secondary antibody (diluted 1:5000).
Cell proliferation was tested by the Cell Counting Kit-8 (CCK-8, Transgene, Beijing, China) according to the manufacturer’s instructions. Cells were first transfected with the miRNAs or plasmids 24 hours in 6-well plates and then reseeded into 96-well plates for testing. 3×103 cells per well were seeded and the cell proliferation was detected 48 hours after transfection.
Statistical analyses were performed with GraphPad Prism 5.0 software. Two-tailed P < 0.05 was considered to be statistically significant.
Cell proliferation was tested by the Cell Counting Kit-8 (CCK-8, Transgene, Beijing, China) according to the manufacturer’s instructions. Cells were first transfected with the miRNAs or plasmids 24 hours in 6-well plates and then reseeded into 96-well plates for testing. 3×103 cells per well were seeded and the cell proliferation was detected 48 hours after transfection.
Statistical analyses were performed with GraphPad Prism 5.0 software. Two-tailed P < 0.05 was considered to be statistically significant.
3
Western Blot Analysis of CLC3 in LNCaP Cells
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LNCaP cells were lysed with radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China), and 30 mg (50 μg/μl) of protein was loaded onto SDS–PAGE gels (EpiZyme Biotech, Shanghai, China) for each sample and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (0.45 mm, Millipore, Bedford, MA). The membranes were blocked at room temperature for 2 hours in 5% evaporated milk dissolved in tris buffered saline‐Tween (TBST) (0.1% Tween‐20, Sigma‐Aldrich, St Louis, MO). Membranes were incubated overnight at 4°C with primary antibodies, including rabbit anti‐CLC3 (1:100, 13359S, Cell Signaling, Danvers, MA) and rabbit anti‐GAPDH (1:750, D16H11, Cell Signaling, Danvers, MA). The secondary horseradish peroxidase (HRP)‐conjugated antibody was goat anti‐rabbit IgG (H + L) (SA00001‐2, Proteintech, Wuhan, China). The bands were detected with Western Blot Luminol Reagent (Santa Cruz, CA).
4
Protein Expression Analysis of UCMSCs
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After cultivation for 7 days, the UCMSCs in the tissue culture plates (TCPs), GelMA-O5 and GelMA-O5/rGO hydrogels were washed with PBS. Then the cells on the gels were treated with liquid nitrogen and smashed. The protein samples were extracted using radioimmunoprecipitation assay (RIPA) protein extraction solution supplemented with protease on the ice for 30 min. The extracted proteins were determined by BCA protein assay (Beyotime, Jiangsu, China). The proteins (20 μg/lane) were resolved by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then the separated proteins were transferred to polyvinylidinedifluoride (PVDF) membranes (0.45 mm; Millipore, Bedford, MA). After blocking with 5% milk for 2 h at room temperature, the membranes were incubated antibodies against rabbit anti-cTnI (ab47003, abcam, 1: 100) and rabbit anti- Cx43 (ab11370, abcam, 1: 100) overnight at 4 °C. After being rinsed for three times with Tris buffered solution (TBST), the membrane was incubated in goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Densitometric analysis was determined using Image J analysis software and β-actin was used as an internal standard.
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