We used previously described methods for the extraction of protein and immunoblotting analyses (Helsley et al., 2016 (link); Sui et al., 2021 (link)). The denatured protein lysates were resolved using SDS-PAGE. The separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, 1620115). After blocking with 5 % bovine serum albumin (BSA, Sigma-Aldrich, A9647) for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C: anti-Actin (1:5000 dilution, Millipore Sigma A2066), anti-Akt (1:1000 dilution, Cell Signaling 9272), and anti-Phospho-Akt (Ser473) antibodies (1:1000 dilution, Cell Signaling 9271). After 4 × 5 min washes with 1 × PBS with 0.1 % Tween20 (PBST), the membranes were incubated for 1 h with an anti-rabbit secondary antibodies (1:5000 dilution, Millipore Sigma 12–348) at room temperature. After 4 × 5 min washes, the membranes were developed by Pierce ECL Western Blotting Substrate kit (Thermo Fisher Scientific, 32209), and exposed with Bio-Rad Chemidoc imaging machine. Uncropped immunobloting images are included in Supplemental Figure 1 .
Chemidoc imaging machine
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc imaging machine is a versatile instrument designed for capturing and analyzing images of various biological samples, such as gels, blots, and plates. The core function of the ChemiDoc is to provide high-quality, reproducible images of these samples, which can then be used for further analysis and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti actin, by Merck Group (2 mentions)
Pierce ecl western blotting substrate kit, by Thermo Fisher Scientific (2 mentions)
Chemiluminescence reagent, by Bio-Rad (2 mentions)
Bca protein kit, by Beyotime (1 mentions)
Tgf β, by Cell Signaling Technology (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ab137332, by Abcam (1 mentions)
Ab179515, by Abcam (1 mentions)
Ab207802, by Abcam (1 mentions)
Ab214185, by Abcam (1 mentions)
Ab215203, by Abcam (1 mentions)
Ab51072, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Ab190183, by Abcam (1 mentions)
5 protocols using chemidoc imaging machine
1
Protein Extraction and Immunoblotting Protocol
2
Western Blot Analysis of Cartilage Proteins
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Western blot was performed following steps previously described [22 (link)]. Briefly, proteins were isolated from cartilage tissues of experimental rats or chondrocytes with RIPA lysis buffer and quantified using BCA protein kit (Both from Beyotime). 10 µg protein/lane was separated by 10% SDS-PAGE and transferred onto PVDF membranes. Subsequently, the membranes were incubated with primary antibodies against USP25, NLRP3, GSDMD-N, active caspase-1, TXNIP, and GAPDH at 4 °C overnight, followed by an incubation with horseradish peroxidase-conjugated secondary antibody for 1 h. Membranes were visualized with chemiluminescence reagent (Bio-Rad) with a ChemiDoc imaging machine (Bio-Rad).
3
Protein Expression Analysis by Western Blot
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Total proteins were isolated from cells by using RIPA lysis buffer containing protease cocktail and PMSF. After quantification, 25μg protein was loaded into the 10% SDS-PAGE gel and separated under electrophoresis. Then the separated proteins were transferred onto PVDF membrane. After blocking by 5% non-fat milk, the membrane was incubated with different primary antibodies including Vimentin (#49636, 1:1000, Cell Signaling Technology), E-cadherin (#14472, 1:1000, Cell Signaling Technology), TGF-β ( #3709, 1:1000, Cell Signaling Technology), p-SMAD2 (#3104, 1:1000, Cell Signaling Technology) and SMAD2 (#5339, 1:1000, Cell Signaling Technology), followed by the HRP-conjugated secondary antibodies (1:5000, Cell Signaling Technology). ChemiDoc imaging machine (Bio-Rad, Berkeley, USA) was used to visualize target proteins with ECL assay.
4
Protein Expression Analysis Protocol
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Protein expression levels in supernatants and whole-cell lysates were measured as previously described (Palazón-Riquelme et al., 2018 (link)). After boiling at 93°C for 5 min, 30 µm of proteins were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membrane. The member was incubated with primary antibodies against USP7 (ab190183), NLRP3 (ab214185), NLRP3 (ab263899), NLRP6 (ab58705), NLRC4 (ab99860), AIM2 (ab233033), GSDMD-N (ab215203), pro-caspase-1 (ab179515), active caspase-1 p20 (ab207802), NOX1 (ab55831), NOX2 (ab129068), NOX3 (ab254572), NOX4 (ab133303), NOX5 (ab198213), MMP1 (ab137332), MMP13 (ab51072; all from Abcam), ASC (orb223237), NLRP1 (orb325922; both from Biorbyt LLC, St Louis, MO, United States), β-catenin (#8480), and GAPDH (#5174; both from Cell Signaling Technology, Danvers, MA, United States) at 4°C for 12 h, and incubated with horseradish peroxidase-conjugated secondary antibody (Beyotime Biotechnology, Shanghai, China; A0208) for 1 h at 22°C after blocking with 3% bovine serum albumin (BSA) for 1 h at 22°C. Membranes were visualized with chemiluminescence reagent (Bio-Rad) with a ChemiDoc imaging machine (Bio-Rad).
5
Protein Extraction and Immunoblotting Protocol
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