The IHC was performed using conventional protocols which are available elsewhere. The primary antibodies were anti-c-PARP (Abcam, Hong Kong, China, #ab32064), anti-HMGB1 (Abcam, #ab18256), anti-GPX4 (Abcam, #ab125066), anti-HBA1 (Abcam, #ab191183), anti-STMN1 (Abcam, #ab52630), anti-HIC1 (Abcam, #ab235037) and anti-HNF4A (R&D Systems, Wiesbaden, Germany, PP-H6939-00 and Abcam, #ab181604). The tissue microarray assay (TMA) slides used in this study were purchased from U.S. Biomax agented by Alenabio (Xi'an, China) and OUTDO Biotech Co. LTD (Shanghai, China). Immunohistochemistry staining was assessed by independent pathologists.
Ab181604
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab181604 is a laboratory product manufactured by Abcam. It is a scientific instrument designed for use in research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Ab125066, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Ab18256, by Abcam (1 mentions)
Ab191183, by Abcam (1 mentions)
Ab52630, by Abcam (1 mentions)
Pp h6939 00, by Abcam (1 mentions)
Ab10297, by Abcam (1 mentions)
Ab188408, by Abcam (1 mentions)
Ab9106, by Abcam (1 mentions)
Ab40084, by Abcam (1 mentions)
Ab104836, by Abcam (1 mentions)
Ab124824, by Abcam (1 mentions)
Ab69644, by Abcam (1 mentions)
Ab19256, by Abcam (1 mentions)
Ab16898, by Abcam (1 mentions)
Ab129058, by Abcam (1 mentions)
Ab5103, by Abcam (1 mentions)
Cell lysis buffer, by Promega (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab9193, by Abcam (1 mentions)
13 protocols using ab181604
1
Immunohistochemical Analysis of Apoptosis Markers
2
Western Blot Analysis of Cellular Proteins
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Tissue or cellular protein was extracted with 1 × cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described [9 (link), 11 (link)–15 (link)], with antibodies for HNF4A (ab181604), hexokinase 2 (HK2, ab104836), solute carrier family 2 member 1 (SLC2A1, ab40084), v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN, ab16898), hnRNPU (ab10297), CTCF (ab188408), clusterin (CLU, ab69644), C-X-C motif chemokine receptor 4 (CXCR4, ab124824), trophoblast glycoprotein (TPBG, ab129058), uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA, ab99323), FLAG (ab125243), Myc (ab9106), glutathione S-transferase (GST, ab19256), histone H3 (ab5103), or β-actin (ab6276, Abcam Inc., Cambridge, MA).
3
Antibody and Inhibitor Inventory
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Horse anti-HBs (ab9193), rabbit anti-HBx (ab39716), mouse anti-HNF4α (ab181604), rabbit anti-actin (ab179467), and HRP-linked rabbit anti-horse IgG (ab6921) antibodies were purchased from Abcam (Cambridge, MA, USA). The rabbit anti-preS1 (10R-10460) antibody was purchased from Fitzgerald (Acton, MA, USA). The HRP-linked goat anti-rabbit IgG (BE0101-100) and HRP-linked goat anti-mouse IgG (BE0102-100) antibodies were purchased from Easybio (Beijing, China). The proteasome inhibitor MG132 (S2619) and lysosome inhibitor HCQ (S4430) were purchased from Selleck (Houston, TX, USA).
4
HNF4α Protein Interactions
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For the co-IP experiments, HPAEpiCs were lysed on ice for 30 min in cell lysis buffer (P0013, Beyotime, Shanghai, China). After centrifugation at 12,000 rpm for 30 min at 4 °C, the supernatant was collected and incubated with anti-HNF4α antibodies (ab181604, 1:70, Abcam, Shanghai, China) overnight. After 4 hr of incubation with Protein A Agarose (20333, Thermo Scientific, Shanghai, China) at 4 °C, the complexes were washed three times. Immunoblotting was performed after elution.
5
SP1 and HNF4α Chromatin Immunoprecipitation
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ChIP was performed as described previously (Tao et al., 2016 (link)) using a ChIP assay kit (P2078, Beyotime) following the manufacturer’s directions. After crosslinking with formaldehyde, the chromatin solutions were sonicated and incubated with anti-SP1 (9389, 1:100, Cell Signaling Technology) and anti-HNF4α (ab181604, 1:100, Abcam) antibodies and control IgG, then rotated overnight at 4 °C, respectively. After purification using a DNA purification kit (BioTeke Corp.), the immunoprecipitated DNA was detected for PCR analysis. All ChIP-qPCR experiments were performed with three biological replicates.
6
Chromatin Immunoprecipitation of HNF4A
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After crosslinking, 5 × 105 chromatin samples were broken for immunoprecipitation with anti‐HNF4A antibody (ab181604, Abcam) or control anti‐IgG antibody (ab172730, Abcam). The antibodies used herein were all chromatin immunoprecipitation (ChIP) Grade. The sample was subjected to 14 times of sonication cycles every 9 sec. Followed by incubation with Protein A/G mix magnetic beads for an hour, precipitated samples were collected for qRT‐PCR analysis.
7
Western Blot Analysis of Protein Expression
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Protein from cells or mouse tissues was extracted by RIPA lysis buffer (65 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) as previous described 36 (link). Protein samples were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (IPVH00010, Merck Millipore, CA, USA). The expression of protein was detected by a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Primary antibodies used were: PRMT1 (1:1000, #07-404, Millipore), β-actin (1:1000, #8457, CST), Flag (1:1000, 2368, CST), PGC-1α (1:2000, ab54481, Abcam), HNF-4α (1:1000, ab181604, Abcam), CD11b (1:1000, ab133357, Abcam) and albumin (1:1000, ab207327, Abcam).
8
ChIP Assay for HNF4α Binding
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ChIP experiments were performed using the EZ-Magna ChIP kit according to the manufacturer's instructions. Briefly, primary hepatocytes in a 150 mm culture dish were treated with vehicle or PCN for 48 h and fixed by adding formaldehyde to the medium. Cells were harvested with lysis buffer and nuclear lysates were sonicated to yield DNA fragments of 0.2–1 kb. IP was performed using antibody to HNF4α (ab181604; Abcam, Cambridge, UK) or rabbit IgG. The purified DNA was amplified by real-time PCR. The primers used in the ChIP assays are as follows: Glut2 gene's promoter was 5ʹ-TCCTTCCGACCTTCATGTTC-3ʹ and 5ʹ-GCTGTGCATTGGAGACTCAC-3ʹ.
9
HNF4α Binding Analysis in HepG2 Cells
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EMSA and antibody‐supershift assay were performed using Chemiluminescent EMSA Kit (Beyotime, Shanghai, China). In brief, the biotin‐labeled probes were incubated with nuclear proteins of HepG2 cells for 30 min at room temperature (RT), followed by native‐PAGE. For competition assay, nuclear proteins were pre‐incubated with an unlabeled consensus binding sequence of HNF4α before labeled probes were added. For antibody‐supershift assay, nuclear proteins were pre‐incubated with anti‐HNF4α antibody (ab181604, Abcam, Cambridge, UK) or isotype‐matched control IgG prior to the addition of labeled probe.
10
Signaling Pathway Immunoblot Protocol
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Antibodies for G6PC (Abcam, Cambridge, MA, USA; ab83690; used at a dilution of 1/500), HNF4α (Abcam, ab181604; used at a dilution of 1/1000), PEPCK (Abcam, ab70358; used at a dilution of 1/1000), and beta-actin (ACTB; Cell Signaling, 4967; used at a dilution of 1/5000) were purchased. Plasmids expressing human H19 (pH19) and empty vector (Vec) were previously described.27 (link) Metformin (ENZO Life Sciences International Inc., Uniondale, NY, USA; ALX-270-432-G005) and DEA (Santa Cruz, Dallas, TX, USA; sc-207632) were purchased. DEA was used at a final concentration of 20 μM.
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