Cells were lysed in RIPA lysis buffer containing protease inhibitors (Thermo Fisher Scientific). Cell protein lysates were centrifuged at 14,000 rpm for 20 min at 4°C and the supernatants were collected. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein from samples were resolved on 4–20% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE; Applygen Technologies) and transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking for nonspecific binding with 5% non-fat dry milk for 1 h at room temperature, the membranes were incubated with antibody HNF1α (1:500, ab96777, Abcam, Cambridge, MA, USA), SCAP (1:2000, ab125186, Abcam), SREBP-2 (1:500, ab30682, Abcam), PCSK9 (1:1000, ab181142, Abcam), or β-actin (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C and followed by an incubation with secondary antibody (1:3000, #7074, Cell Signaling Technology) for 1 h at room temperature. Protein was visualized with enhanced chemiluminescence (ECL; Thermo Fisher Scientific) and membranes were imaged with GE Healthcare imaging system (Waukesha, WI, USA). Band intensities were quantified using Image J software.
Ab96777
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab96777 is a laboratory equipment product offered by Abcam. This product serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab30682, by Abcam (1 mentions)
Ab181142, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab7028, by Abcam (1 mentions)
Ab7030, by Abcam (1 mentions)
Ab28146, by Abcam (1 mentions)
Ab12169, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
Anti kim 1 af1817, by R&D Systems (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Ab150077, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Envision detection rabbit mouse kit, by Gene Tech (1 mentions)
Ab2020, by Abcam (1 mentions)
Sc 101752, by Santa Cruz Biotechnology (1 mentions)
Bm0002, by Boster Bio (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
8 protocols using ab96777
1
Western Blot Analysis of Cholesterol Regulators
2
Romidepsin and Lipopolysaccharide Interactions
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Romidepsin (FK228) (S3020), was purchased from Shanghai Selleck Chemicals Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) derived from Escherichia coli 055: B5 (L2637) was purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies to acetyl-histone H3 (4499) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-KIM-1 (AF1817) was purchased from R&D Systems (Chengdu, China). Antibodies to CYP2E1 (ab28146), HDAC1 (ab7028), HDAC2 (ab12169), HDAC3 (ab7030), hepatocyte nuclear factor-1 alpha (HNF-1α) (ab96777) and Nrf2 (ab62352) were purchased from Abcam (Cambridge, UK). Anti-GAPDH (TA-08) was purchased from ZSGB Biotechnology (Beijing, China).
3
Immunofluorescent Localization of HNF1α
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Cells were fixed with 4% paraformaldehyde (Sigma, 158127) for 10 min, permeated with 0.5% Triton X-100 in PBS for 20 min at room temperature, and then, the cells were blocked with 5% BSA for 1 h. HNF1α antibody (Abcam, ab96777) was added and incubated overnight at 4°C. Fluorescent secondary antibody (Abcam, ab150077) was added for 2 h at room temperature. Cells were then incubated with DAPI for 10 min at room temperature and observed using a fluorescence microscope (Nikon).
4
Quantitative Liver Fibrosis Assessment
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Sirius red was used to stain for collagen. Immunohistochemistry was performed on paraffin-embedded liver sections. Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry. Sections were stained with ImmunoCruz™ goat ABC Staining (Sc-2023, Santa Cruz) or EnVision Detection Rabbit/Mouse Kit (GK500710, GeneTech, Shanghai, China) and counterstained with hematoxylin. Areas of positive stained sites were measured using image analyses software Image-Pro Plus 6.0 (Media Cybernetics). Percentage of positive area in corresponding field of liver tissue was calculated to show the intensity of collagen deposition or protein expression.
5
Western Blot Analysis of Protein Expression
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Proteins were extracted using RIPA buffer (Beyotime Institute of Biotechnology). Protein concentration was measured with the bicinchoninic acid assay (Beyotime Institute of Biotechnology). Following denaturation, 10 µg protein/lane was separated by 10% SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked in 5% non-fat milk for 2 h at room temperature. The membranes were incubated with primary antibodies against HNF1A (1:1000; ab96777; Abcam), DNAJB12 (1:1000; ab154410; Abcam), PCNA (1:1000; ab92552; Abcam), E-cadherin (1:1000; ab231303; Abcam), vimentin (1:1000; ab16700; Abcam) and GAPDH (1:1000; sc-47724; Santa Cruz Biotechnology) overnight at 4°C. Following primary incubation, membranes were incubated with secondary antibodies for 2 h at room temperature. Protein bands were visualized using the Pierce ECL Western Blotting kit (Pierce; Thermo Fisher Scientific, Inc.). Protein expression was quantified using Image-Pro® Plus software (version 6.0; Media Cybernetics, Inc.). GAPDH was used as an endogenous control for data normalization.
6
Western Blot Analysis of Key Proteins
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Western blot assay was performed as described previously [23 (link)]. Primary antibodies were rabbit anti-human HNF1A antibody (1:1000, #ab96777, Abcam), rabbit anti-human ABCB1 antibody (1:1000, #ab170904, Abcam), rabbit anti-human ABCC1 antibody (1:1000, #ab32574, Abcam), rabbit anti-human ABCC3 antibody (1:1000, #ab226804, Abcam), rabbit anti-human ABCC5 antibody (1:1000, #ab24107, Abcam) and rabbit anti-human GAPDH antibody (1:1000, #ab18162, Abcam). They were then incubated with the following HRP-linked secondary antibody: goat anti-rabbit IgG (1: 10000; Cell Signaling Technology, Boston, USA). The specificity of the antibody ab96777 was shown as full western blots of the whole cell lysates (Fig. S1a).
7
Western Blot Analysis of Cellular Proteins
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Tissues and cells were lysed in lysis buffer (125 mM Tris-HCl pH 6.8, 25% glycerol, 5% SDS) supplemented with protease inhibitor (Roche, Switzerland). Lysates were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to methanol-activated nitrocellulose membranes (HAHY00010; Millipore, Merck KGaA, Darmstadt, Germany). Membranes were blocked in PBS containing 1% Tween-20 and 5% milk for 1 h and incubated with primary antibodies overnight at 4°C. After 1 h of incubation with donkey anti-mouse or donkey anti-rabbit secondary antibodies (IRDye 800; LI-COR Biosciences, Lincoln, NE, United States), signals were imaged using an Odyssey infrared imaging system (LI-COR Biosciences) at a wavelength of 700/800 nm. The primary antibodies used were: SREBP1 (ab96777, Abcam; sc-557036, Santacruz), phospho-Akt (4060, Cell Signaling Technologies), Akt (40D4, Cell Signaling Technologies), and GAPDH (YM3029, Immunoway).
8
Immunohistochemical Analysis of Epigenetic Regulators
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Immunohistochemistry was performed on paraffin-embedded EDTA-decalcified knee sections. Heat induced epitope retrieval was performed using a Citrate-EDTA buffer (pH 6.2) for 10 min at 98 C, except for the detection of DOT1L. Sections were treated with 3% H 2 O 2 /methanol for 10 min to inactivate endogenous peroxidase, blocked in goat serum for 30 min and incubated overnight at 4 C with primary antibodies against DOT1L (Abcam, ab64077), COLX (Abcam, ab58632), TCF1 (Abcam, ab96777) or for 90 min with primary antibody against H3K79me2 (Abcam, ab3594). Rabbit IgG (Santa Cruz, sc-2027) was used as negative control. Avidin-biotin complex amplification (Vectastain ABC kit, Vector Laboratories, USA) was used, except for the immunohistochemical detection of DOT1L and H3K79me2. Peroxidase goat anti-rabbit IgG (Jackson Immunoresearch, Suffolk, UK) was applied for 30 min and peroxidase activity was determined using DAB. For detection of COLX, antigen retrieval was performed as reported 12 . Quantification of the DAB staining was performed with color deconvolution plugin (Jacqui Ross, Auckland University) in ImageJ Software (NIH Image, National Institutes of Health). Quantification was performed using two technical replicates for three different mice per condition in separate experiments 22 , with staining intensity reported relative to the control mice in the same experiment.
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