Chromatin samples for ChIP were prepared using a Simple ChIP Enzymatic Chromatin IP Kit (CST) and were precleaned with Protein A-conjugated Dynabeads (Thermo Fisher Scientific) at 4°C for 2 h. Antibodies against Oct-1 (ab15112, Abcam) and Oct-2 (ab179808, Abcam) were bound to Protein A-conjugated Dynabeads, then subjected to immunoprecipitation with the precleaned samples at 4°C overnight. Washing beads and eluting immunoprecipitated DNA fragments were prepared according to the CST manual. qPCR was performed as described above. The qPCR primer sequences were determined based on the Oct-1 or Oct-2 binding sites deposited in the JASPAR and DECODE databases. The qPCR primer sequences for ChIP-PCR are listed in Table 3 . Data were normalized using the Ct values of input samples.
Ab179808
Manufactured by Abcam
Sourced in United Kingdom
Ab179808 is a protein that functions as a cell surface receptor. It is commonly used in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Protein a conjugated dynabeads, by Thermo Fisher Scientific (1 mentions)
Ab140601, by Abcam (1 mentions)
Ab179434, by Abcam (1 mentions)
Magne halotag beads, by Promega (1 mentions)
Bp clonase 2, by Thermo Fisher Scientific (1 mentions)
Pcdna3.2 v5 dest, by Thermo Fisher Scientific (1 mentions)
Ha tagged ubiquitin plasmid, by Addgene (1 mentions)
Halotev protease, by Promega (1 mentions)
Hc201 01, by Transgene (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Dmi3000b inverted fluorescence microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Monosaccharide standards, by Yuanye Bio-Technology (1 mentions)
Ab260038, by Abcam (1 mentions)
Ab15603, by Abcam (1 mentions)
7 protocols using ab179808
1
Chromatin Immunoprecipitation for Oct-1 and Oct-2
2
Investigating USP2A Interactions with Transcription Factors
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The coding region of human USP2A (accession number NM_004205) without the stop codon was cloned into pcDNA3.2/V5-DEST (Thermo Fisher Scientific) using BP clonase II (Thermo Fisher Scientific). The resultant plasmid encoded C-terminal V5-tagged USP2A. Halo-tagged Oct-1 and Oct-2 expression plasmids were purchased from Promega (Madison, WI). The HA-tagged ubiquitin plasmid was purchased from Addgene (Cambridge, MA). The USP2A, or control pcDNA3.2/V5-DEST plasmid, plasmids encoding Halo-tagged Oct-1 or Oct-2 and HA-tagged ubiquitin were transfected into HEK293FT cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). The pull-down and elution of Oct proteins were performed using Magne HaloTag beads (Promega) and HaloTEV protease (Promega) as per the manufacturer's instructions. For detection of Western blot bands, corresponding to K48- and K63-linked ubiquitin chains, Oct proteins, HA-tagged ubiquitin, and V5-tagged USP2, 1000-fold-diluted antibodies against polyubiquitin chains formed by K48 residues (ab140601; Abcam) and K63 residues (ab179434; Abcam), Oct-1 (A301-717A; Bethyl Laboratories), Oct-2 (ab179808; Abcam), HA-tag (#3724; CST), and V5-tag (A190-120F; Bethyl Laboratories) were used as the primary antibodies.
3
Kidney Protein Expression Analysis
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Kidney tissues were collected and lysed with Mammalian Protein Extraction Reagent, supplemented with protease inhibitor and phosphatase inhibitor, on ice for 45 min. Subsequently, all lysate was mixed with loading buffer and boiled at 98 °C for 8 min. The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Then, the membranes were incubated with primary antibodies organic cation transporter 2 (Oct2) (ab179808, Abcam, Cambridge, UK) and B-actin (HC201-01, Transgene, Beijing, China) overnight at 4 °C, and secondary antibodies for 1 h. ImageJ software was used to quantitatively analyze the expression of proteins.
4
Investigating POU2F2 and AGO1 in Cancer
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Anti-POU2F2 (1:200 for Immunohistochemical (IHC), 1:1000 for Immunoblot, and 1:50 for CHIP, ab179808, Abcam, UK), anti-AGO1 (1:100, ab129304, Abcam, UK), anti-Ki67 (1:1000, ab16667, Abcam, UK), anti-PCNA (1:1000, ab29, Abcam, UK), anti-N-cadherin (1:500, ab76011, Abcam, UK), and anti-β-actin (1:1000, ab8226, Abcam,UK). POU2F2 and AGO1 siRNAs were bought from Riobio Biotech. The shRNA of POU2F2, plv-vector, plv-POU2F2, plv-AGO1, and pGL3-Basic plasmids was purchased from Addgene plc.
5
Quantitative Protein Expression Analysis
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Proteins were separated by 10% SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blotted with 5% skimmed milk and subsequently probed overnight at 4°C with primary antibodies specific for Oct1 (Slc22a1, ab66132), Oct2 (Slc22a2, ab179808), Mate1 (Slc47a1, ab105049), Mate2 (Slc47a2, ab174344) and Gapdh (ab8245; Abcam, Cambridge, UK) and then incubated with horseradish peroxidase conjugated goat anti‐rabbit or antimouse secondary antibody. After washing with TBST buffer, the membranes were developed using enhanced chemilu‐minescent reagent and subjected to autoluminography for 1–5 min.
6
Immunostaining and Inhibition Assay Protocol
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For immunostaining, cells were seeded onto a coverslip prior to infection. Infected cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS. Cells were then incubated with the primary antibodies for 4 h at 37 C. The primary antibodies rabbit anti-mCAT-1 (MBS710656, Santa Cruz Biotechnology) or anti-Oct-2 (ab179808, Abcam) were diluted 1:100 in PBS containing 1% bovine serum albumin, 1% goat serum, and 0.1% saponin. Cells were washed with PBS, followed by incubation with rhodamine red-conjugated anti-rabbit IgG (R-6394, Thermo Fisher Scientific) for 1 h at 37 C. Samples were viewed on a Leica DMI3000B inverted fluorescence microscope. Nuclei were stained with DAPI (C1005, Beyotime Biotechnology).
mCAT-1/Oct-2 inhibition assay nlrc4 À/À BMDMs were primed with LPS (250 ng/ml) for 3 h, followed pretreatment mCAT-1 inhibitor (Cyclosporine, 3 mg/ml, Selleck, Selleck Chemicals) or Oct-2 inhibitor (Cimetidine 200 mM, Selleck, Selleck Chemicals) for 1 h. Then infected with E. piscicida at indicated time points for analysis.
mCAT-1/Oct-2 inhibition assay nlrc4 À/À BMDMs were primed with LPS (250 ng/ml) for 3 h, followed pretreatment mCAT-1 inhibitor (Cyclosporine, 3 mg/ml, Selleck, Selleck Chemicals) or Oct-2 inhibitor (Cimetidine 200 mM, Selleck, Selleck Chemicals) for 1 h. Then infected with E. piscicida at indicated time points for analysis.
7
Investigating Oxaliplatin Resistance in A. chinensis Root Extracts
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The roots
of A. chinensis (DC.) Koidz. (210301)
were purchased from Guangzhou Zisun Pharmaceutical Co., Ltd. (Guangzhou,
China) and authenticated by Nengfeng Ou, the pharmacist in charge
of assessing herb quality at the Second Affiliated Hospital of Guangzhou
University of Chinese Medicine. MKN45 cells were purchased from the
China Center for Type Culture Collection (Wuhan, China). Oxaliplatin
(20220223) was purchased from Jiangsu Hengrui Pharmaceutical Co.,
Ltd. (Chengdu, China). RIPA lysis buffer (P0013B) was purchased from
Beyotime Biotechnology Co., Ltd. (Shanghai China). The monosaccharide
standards were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai,
China). Dextran standards were purchased from the American Polymers
Standards Corporation (USA). Antibodies against OCT2 (ab179808), MRP1
(ab260038), and MRP2 (ab15603) were purchased from Abcam (Cambridge,
UK). GAPDH (D16H11, CST, Danvers, MA) and Pt ICP-MS standard solution
(060078-04-01) were purchased from O2si Smart Solutions (North Charleston,
USA).
of A. chinensis (DC.) Koidz. (210301)
were purchased from Guangzhou Zisun Pharmaceutical Co., Ltd. (Guangzhou,
China) and authenticated by Nengfeng Ou, the pharmacist in charge
of assessing herb quality at the Second Affiliated Hospital of Guangzhou
University of Chinese Medicine. MKN45 cells were purchased from the
China Center for Type Culture Collection (Wuhan, China). Oxaliplatin
(20220223) was purchased from Jiangsu Hengrui Pharmaceutical Co.,
Ltd. (Chengdu, China). RIPA lysis buffer (P0013B) was purchased from
Beyotime Biotechnology Co., Ltd. (Shanghai China). The monosaccharide
standards were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai,
China). Dextran standards were purchased from the American Polymers
Standards Corporation (USA). Antibodies against OCT2 (ab179808), MRP1
(ab260038), and MRP2 (ab15603) were purchased from Abcam (Cambridge,
UK). GAPDH (D16H11, CST, Danvers, MA) and Pt ICP-MS standard solution
(060078-04-01) were purchased from O2si Smart Solutions (North Charleston,
USA).
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