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).
Sqstm1 p62
Manufactured by Beyotime
Sourced in China
SQSTM1/p62 is a protein that functions as a receptor for polyubiquitinated proteins, facilitating their delivery to the proteasome for degradation. It acts as a link between ubiquitinated proteins and the autophagic machinery.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Beyotime (2 mentions)
Cyclin b1, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by ZenBio (2 mentions)
Caspase 3, by ZenBio (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Cytochrome c, by Proteintech (1 mentions)
Tom20, by Beyotime (1 mentions)
Sars cov 2 np, by Sino Biological (1 mentions)
Flag tag (flag), by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Tim23, by Proteintech (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by CWBIO (1 mentions)
Parp1, by ZenBio (1 mentions)
Caspase 9 p35 p10, by Proteintech (1 mentions)
Ncm460, by Hunan Fenghui Biotechnology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Hct116, by Procell (1 mentions)
Bcl 2, by Abmart (1 mentions)
4 protocols using sqstm1 p62
1
SARS-CoV-2 Protein Localization and Interaction
2
Investigating Colon Cancer Cell Lines
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Human colon cancer cell lines (HCT116 and DLD1) were purchased from Procell Company (Wuhan, China) and human colon mucosal epithelial cell line (NCM460) were purchased from Fenghui Biotechnology (Hunan, China). All cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 100 U/mL mixture of penicillin and streptomycin. The TDA is provided by the School of Pharmacy, Chongqing University. Cell counting kit 8 was purchased from MCE. Annexin V-FITC/PI Apoptosis Detection kit was purchased from CWBIO (Beijing, China). Primary antibodies for Western blotting, immunofluorescence and immunohistochemistry against target proteins are shown as follows: Caspase9/P35/P10 (Proteintech, Chicago, IL, USA, 66169), Caspase3 (Zen Bioscience, Chengdu, China, 300968), cleaved-Caspase3 (Zen Bioscience, 380169), PARP1 (Zen Bioscience, 380451), LC3 (Sangon Biotech, Shanghai, China, D163557), ATG5 (Sangon Biotech, D121650),SQSTM1/p62 (Beyotime, Shanghai, China, AF5312), β-actin (Santa Cruz, sc-47778), cyclinD1 (Cell Signaling Technology, USA, 2978S), cyclinB1 (Cell Signaling Technology, 4135S), PERK (Cell Signaling Technology, 3192S), IRE1 (Zen Bioscience, 220399), XBP1(Zen Bioscience, 381710), CHOP (Zen Bioscience, 381679) and HSPA5 (Sangon Biotech, D260466).
3
Western Blot Analysis of Apoptosis-Related Proteins
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Total proteins were loaded by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk. Membranes were incubated with the following primary antibodies at 4°C overnight: GPX-4 (#bs-3884R; Bioss, China), B-cell lymphoma 2 (Bcl-2; # T40056; Abmart, China), Bcl-2-associated X protein (Bax; #T40051; Abmart, China), cleaved caspase-3 (#9644T; Cell signaling technology, USA), DNA damage-inducible transcript 3 protein/enhancer-binding protein homologous protein (DDIT3/CHOP; #AF6684; Beyotime Biotechnology, China), Beclin 1 (#AF5123; Beyotime Biotechnology, China), SQSTM1/p62 (#AF5312; Beyotime Biotechnology, China), ATG5 (#T55766; Abmart, China), and LC3B (#T55992; Abmart, China). A digital gel image analysis system Amersham Imager 600 (GE Healthcare Bio-Sciences, USA) was used to measure and analyze the density of the bands.
4
Cyclovirobuxine D Induces Lung Cancer Cell Apoptosis
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Human lung cancer cell lines (A549, H446 and 95-D) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM containing 10% fetal bovine serum (FBS) and a 100 U/mL mixture of penicillin and streptomycin. All cells were kept at 37 °C and 5% CO2. Cyclovirobuxine D (CVB-D) was purchased from the Chengdu Alpha Biotechnology Incorporation and dissolved in methanol to generate a 70 mM stock solution. The primary antibodies for Western blotting, immunoprecipitation and immunohistochemistry against target proteins are as follows: caspase3 (Zen Bioscience, 300968, Chengdu, China), cleaved-caspase3 (Zen Bioscience, 380169), PARP (Zen Bioscience, 380451), cleaved-PARP (Zen Bioscience, 380374), VDAC1 (Sangon Biotech, D124100, Shanghai, China), TOMM20 (Sangon Biotech, D153158), COX4I1 (Sangon Biotech, D262690), LC3 (Sangon Biotech, D163557), ATG5 (Sangon Biotech, D121650), SQSTM1/p62 (Beyotime, AF5312), BNIP3 (Zen Bioscience, 381756), BNIP3L (Zen Bioscience, 381891), β-actin (Santa Cruz, sc-47778), p65 (Cell Signaling Technology, 8242S, Danvers, MA, USA), CDC2 (Cell Signaling Technology, 9116S), cyclinB1 (Cell Signaling Technology, 4135S), p-BNIP3L (phosphoS81) (abcam, ab208190, Cambridge, UK) and phosphoserine (Millipore, AB1603, Burlington, MA, USA).
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