Proteins from hippocampal tissues or cell lysates (20–40 μg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, and blocked with 5% BSA/Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. Then, the membranes were incubated overnight at 4°C with primary antibodies at the following dilutions: NLRP3 (1:1000; abcam), caspase-1 (1:200; Santa cruz), ASC (1:1000; abcam), Bax (1:1000; CST), Bcl-2 (1:2000; CST), caspase-3 (1:1000; CST), Parkin (1:1000; CST), PINK1 (1:1000; abcam), LC3 (1:1000; CST), ATG5 (1:1000; CST), ATG7 (1:1000; CST), P62 (1:1000; CST), Beclin-1 (1:1000; CST), TOM20 (1:500; Beyotime), and GAPDH (1:500; Beyotime), followed by incubation with appropriate secondary antibodies after thoroughly washing three times with TBST. The bands were visualized using the chemiluminescence (ECL) detection system (Thermo Fisher Scientific) and quantified by Image J gel analysis software. Expression levels were normalized against GAPDH.
Tom20
Manufactured by Beyotime
The TOM20 is a laboratory equipment designed for the analysis and characterization of mitochondrial protein import receptors. It is a key component in the study of mitochondrial biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Pink1, by Abcam (1 mentions)
Caspase 1, by Santa Cruz Biotechnology (1 mentions)
Nlrp3, by Abcam (1 mentions)
Gapdh, by Beyotime (1 mentions)
Cytochrome c, by Proteintech (1 mentions)
Flag tag (flag), by Proteintech (1 mentions)
Sqstm1 p62, by Beyotime (1 mentions)
Sars cov 2 np, by Sino Biological (1 mentions)
Beclin 1, by Beyotime (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Tim23, by Proteintech (1 mentions)
2 protocols using tom20
1
Western Blot Analysis of Hippocampal Proteins
2
SARS-CoV-2 Protein Localization and Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The Nsp1–10 and Nsp12–16 fragments were obtained from the SARS-CoV-2 (MN908947.3) cDNA provided by Prof. Ke Peng (Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China) (Li et al., 2020 (link)). The Nsp1–10, Nsp12–16, and N- and C-terminal domains of Nsp8 fragments were cloned into pCMV-Flag vector. And the full-lenth, N- and C-terminal domains of Nsp8 were cloned into pCMV-GFP vector to generate pCMV-GFP-Nsp8, pCMV-GFP-Nsp8-N and pCMV-GFP-Nsp8-C plasmids. Flag-Nsp8 was cloned into pCDH-puro-EGFP to generate the pCDH-Nsp8-puro-EGFP plasmid. Supplementary Table S1 lists the primers used in plasmid construction. Mito-DsRed2-EGFP (Cat#P4954) and mCherry-EGFP-LC3 (Cat#P0446) plasmids were purchased from Miaoling Plasmid Sharing Platform (Wuhan, China). EGFP-LC3 was cloned by mutation of mCherry-EGFP-LC3. The following primary antibodies were used: TOM20 (Beyotime, AF1717); SQSTM1/p62 (Beyotime, AF5312); Beclin 1 (Beyotime, AF5123); ATG5 (Beyotime, AF2269); LC3A/B (Cell Signaling Technology, 12741S); anti-Mouse IgG 647 (Invitrogen, A31571); anti-Mouse IgG594 (Invitrogen, A21203); Flag (Proteintech, 20543-1-AP); β-Actin (Proteintech, 66009-1-Ig); Cytochrome c (Proteintech, 10993-1-AP); COXII (Proteintech, 55070-1-AP); TIM23 (Proteintech, 11123-1-AP); SARS-CoV-2-NP (Sino Biological, 40143-MM05).
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