After the treatments, the cells were lysed on ice for 20 min with a mixture of 100 μl RIPA buffer (Fdbio science, Hangzhou, China), 1 μl protease inhibitor (Fdbio science) and 1 μl phosphatase inhibitor (Fdbio science) and centrifuged at 14,000 g for 25 min. Protein concentrations were determined using a BCA Total Protein Assay Kit (CWBIO, China). Protein samples (40 μg/lane) were separated on SDS-PAGE gels (8% or 12%, Epizyme, China) and electrotransferred to PVDF membranes. The blots were incubated with the following primary antibodies: anti-NLRP3 (1:500, A12694, ABclonal), anti-GSDMD (1:500, 39754, CST), anti-p20 (1:500, AF4005, Affinity), anti-CHOP (1:500, ab11419, Abcam), anti-GRP78 (1:500, ab21685, Abcam), anti-COL2A (1:500, ab34712, Abcam), anti-Aggrecan (1:500, ab36861, Abcam), anti-MMP3 (1:500, ab52915, Abcam), anti-MMP13 (1:500, ab39012, Abcam), anti-ADAMTS5 (1:1000, ab41037, Abcam), and anti-SREBP1 (1:1000, ab28481, Abcam). The membranes were washed and incubated with the HRP-conjugated secondary antibodies (1:8000, ABclonal, China) for 1 h. The bands were developed with chemiluminescence reagents and imaged by a myECL imager (Syngene G:BOX ChemiXT4, United Kingdom). The experiment was repeated in triplicate, and the blots were quantified by ImageJ software. An antibody against β-actin (AC026, 1:1000; ABclonal) served as an endogenous control.
Ab28481
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab28481 is a synthetic peptide that can be used as a positive control in immunoassays. The peptide sequence is derived from a human protein and is conjugated to a carrier protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab82419, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab176323, by Abcam (3 mentions)
Ab236868, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab8805, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Ab22759, by Abcam (3 mentions)
Ab30532, by Abcam (2 mentions)
Ab72046, by Abcam (2 mentions)
Imaris viewer, by Oxford Instruments (2 mentions)
Alexa fluor 488, by Cell Signaling Technology (2 mentions)
Ab40793, by Abcam (2 mentions)
Ab3484, by Abcam (2 mentions)
Bs 12867r, by Bioss Antibodies (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab18180, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
44 protocols using ab28481
1
Comprehensive Protein Analysis of Cellular Responses
2
Lipid Metabolism Regulation Assay
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Triacylglycerols (TG), Total cholesterol (TCH), Low-density lipoprotein cholesterol (LDL-C), High-density lipoprotein cholesterol (HDL-C), free fatty acids (FFA) and total bile acid (TBA) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies against liver X receptor-α (LXRα) (ab176323), peroxisome proliferator activated receptor γ (PPARγ) (ab45036), sterol regulatory element binding protein 1c (SREBP-1c) (ab28481), cluster of differentiation 36 (CD36) (ab133625), acyl-CoA carboxylase 1 (ACC1) (ab72046), fatty acid synthase (FAS) (ab15285), low-density lipoprotein receptor (LDLR) (ab30532), diacylglycerol acyltransferase 2 (DGAT2) (ab237613) and Goat Anti-Rabbit IgG H&L (HRP) (ab205718) were purchased from Abcam company(Cambridge, UK).
3
Quantifying Hepatic Lipid and Protein Markers
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The liver tissues initially fixed with 4% paraformaldehyde plus 20% sucrose overnight and sectioned to 8 μm with a cryostat (Thermo Fisher Scientific, MA, USA). For oil red O (ORO) staining, the liver cryosections were stained with ORO to visualize the lipid droplets. Images were collected by Leica automatic digital slide scanner (Aperio VERSA 8, GER) and quantified with ImageJ (National Institutes of Health, USA). Immunofluorescence on the liver cryosections was performed according to a previous study [33 (link)]. Primary antibodies were anti-SREBP1 (Abcam, ab28481, and Rabbit, 1 : 200), anti-P-AKTSer473 (Cell Signaling Technology, 4060, Rabbit, 1 : 200), and anti-CHREBP (Abcam, ab81958, Rabbit, 1 : 200), followed by Alexa Fluor 568 (Thermo Fisher Scientific, A11036, Goat anti rabbit, 1 : 1,000) or Alexa Fluor 488 (Cell Signaling Technology, 4412, Goat anti rabbit, 1 : 1,000) conjugated secondary antibody for visualization. Nuclei were stained by Hoechst 33342 (Thermo Fisher Scientific, H21492). Images were collected with Leica laser-scanning confocal microscope (SP8 DLS, GER), analyzed by Imaris Viewer (Oxford Instruments, UK), and quantified by ImageJ (National Institutes of Health, USA).
4
Immunohistochemical Analysis of Metabolic Enzymes
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Immunohistochemical slides were evaluated by three pathologists (G.B., E.P. and R.C.) with no information on patient clinical data, as previously described [19 (link),20 (link)]. Immunohistochemical analyses were performed according to previous methodology [19 (link)], As concerning new experiments, we used FASN (fatty acid synthase) antibody (C20G5; Rabbit IgG monoclonal, 1:50–1:200 dilution) [21 (link)], Carnitine palmitoyltransferase I (CPT-1) (15184-1-AP, Proteintech, Rosemont, IL, USA, 1:1500 dilution, Benchmark XT I-VIEW DAB detection kit from Ventana, Roche Group, Basel, Switzerland) [22 (link)], Sterol regulatory element-binding protein (SREBP1) (ab28481, rabbit polyclonal antibody, 1:50–1:500 dilution, Abcam, Cambridge, UK) [23 (link)], ATP citrate lyase: anti-ACLY (ab40793, rabbit monoclonal antibody, Abcam) [23 (link)], Stearoyl-CoA desaturase-1 (SCD) (BS-3787R, rabbit polyclonal, 1:400 dilution, Bioscience, Allentown, PA, USA) [24 (link)], Acetyl-CoA Carboxylase 1 (ACC-1) (cat. #4190, rabbit monoclonal, 1:50 dilution, Cell Signaling Technology, Danvers, MA, USA) [25 (link)]. The scoring applied was conducted following previous methodology [19 (link),20 (link),26 (link)].
5
Western Blot Protein Quantification in Liver
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Protein levels were analyzed through Western blot analyses as described previously [45 (link)]. Briefly, snap-frozen livers were homogenized and diluted. Protein content of homogenates was determined with the DC assay kit (Bio-Rad, Munich, Germany). Samples (30 µg of protein/lane) were subjected to SDS-PAGE (10% gels), and proteins were blotted on nitrocellulose membranes. Glyceraldehyde-3-phosphate dehydrogenase served as an endogenous control (sc166545HRP, Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were blocked and incubated with primary antibodies for either fatty acid synthase (ab82419, Abcam, Berlin, Germany), or sterol regulatory element-binding protein 1c (ab28481, Abcam, Berlin, Germany). Finally, membranes were incubated with the corresponding secondary antibody, and blots were developed using enhanced chemiluminescence. Protein quantification was performed by Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan).
6
Western Blot Analysis of Protein Markers
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Proteins were extracted from cells using RIPA buffer (89900, Thermo Fisher Scientific, Grand Island, NY, USA), and protein concentration was measured by using a BCA assay (23225, Thermo Fisher Scientific), and equal quantities of protein were separated by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and blocked with 5% bovine serum albumin. Blocked PVD membranes were incubated overnight at 4°C with primary antibodies at dilutions recommended by the manufacturers. The following primary antibodies were used for western blot analysis: anti-SCD1 (Abcam, ab236868, 1 : 1,000), anti-GAPDH (Abcam, ab8245, 1: 1,000), anti-TSG101 (Abcam, ab83, 1 : 1,000), anti-CD81 (Abcam, ab286173, 1 : 500), anti-CD63 (Abcam, ab1318, 1 : 1,000), anti-HSP70 (Abcam, ab2787, 1 : 1,000), anti-β-actin (Abcam, ab7817, 1 : 2,000), anti-SREBP-1 (Abcam, ab28481, 1 : 1,000), anti-LRP5 (Abcam, ab223203, 1 : 1,000), and anti-LRP6 (Abcam, ab231779, 1 : 1,000). Membranes were then incubated with secondary antibodies for 1 h at room temperature and immunoreactive bands were detected by enhanced chemiluminescence (Thermo Fisher Scientific). The density of the bands was analyzed by ImageJ.
7
Investigating Lipid Metabolism Pathways
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Palmitic acid (PA), stearic acid (SA), and fatty acid-free bovine serum albumin (BSA) were purchased from Sigma–Aldrich. C34, MG132 and actinomycin D were purchased from Caymen Chemicals. Antibodies against SREBP1 (ab28481), MCAD (ab92461), TLR4 (ab13556), TIRAP (ab17218), MyD88 (ab135693), and β-actin (ab6276) were purchased from Abcam. Antibodies against Sp1 (#9389), PU.1 (#2266), ACC (#3662), FASN (#3180), CPT1A (#97361), GLUT1 (#73015), p-NFκB (#3033), and β-catenin (#8480) were purchased from Cell Signaling. Horseradish peroxidase (HRP)-conjugated secondary antibodies (sc2357) were purchased from Santa Cruz Biotechnology. TLR4 promoter reporters were given as a kind gift by Professor Michael Rehli at the University of Regensburg Medical School, Germany. All the diets, including high-fat diet (HFD, D12492) and its matched control diet (CD, D12450J), Palmitic acid-rich diet (PAD, D16042106) and its matched control diet (C-PAD, D17042705), were purchased from Research Diets, Inc.
8
Western Blot Analysis of Insulin Signaling Proteins
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After six weeks of drug therapy, all mice were euthanized by intraperitoneal injection with 0.1% pentobarbital sodium. The adipose tissue from the other three groups of mice were removed and stored at −80°C to examine the protein expressions of Insig-1, SCAP, and SREBP-1c. The protocol for western blotting was previously described by us [17 (link)]. Primary antibodies including Insig-1 (Abcam, ab70784, 1 : 200), SREBP-1c (Abcam, ab28481, 1 : 1000), SCAP (Abcam, ab19013, 1 : 1000), IRS-1 (Abcam, ab52167, 1 : 1000), PI3K (Abcam, ab151549, 1 : 1000), Akt (Abcam, ab8805, 1 : 500), mTORC1(Abcam, ab2732, 1 : 200), PAQR3 (Abcam, ab174327, 1 : 250), and β-actin (Beijing Zhongshan Golden Bridge Biotechnology Co., TA-09, 1 : 1000) were used in this study. Protein expression was detected with an enhanced chemiluminescence detection system (Vigorous, Beijing, China).
9
Cholesterol Trafficking and Lysosomal Analysis
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Chemical and standard reagents including filipin and NBD-cholesterol were obtained from Sigma-Aldrich, St. Louis, MO. LysoTracker and fluorescent dextran were obtained from Life Technologies (Grand Island, NY). shRNA constructs were obtained from SA Biosciences (Valencia, CA). Antibodies against tubulin [1:100 for immunofluorescence (IF), T6199, Sigma-Aldrich], EGFP (1:100 for IF, 632380, Clontech) and mCherry (1:100 for IF, NBP1-96752, Novus Biologicals) have been described previously (Whyte et al., 2008 (link); Towns et al., 2009 (link)). Antibodies against StARD9 [1:100 for western blotting (WB), HPA014562] were obtained from Sigma-Aldrich. Site-directed mutagenesis utilized the Phusion kit (New England Biolabs, Ipswich, MA). Antibodies against NPC1 (5 μg/ml for IF, 1 μg/ml for WB, ab108921), LAMP1 (1:10 for IF, ab25630), SREBP1 (1:100 for IF, ab28481) and cathepsin B (1 μg/ml for IF, ab6313) were obtained from Abcam (Boston, MA). filipin (f9765) was obtained from Sigma-Aldrich. NBD-cholesterol (N1148) was obtained from Invitrogen (Waltham, MA). BacMAM-ER (C10590) was obtained from Thermo Fisher Scientific (Waltham, MA). Nocodazole (M1404) and taxol (T7402) were obtained from Sigma-Aldrich. The sequences of oligonucleotides used in this study are provided in Table S3 .
10
Protein Expression Analysis Protocol
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The total protein isolation, denaturing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferring, blocking, incubation, visualization, and quantification assay were based on our published methods (21 (link)). The following antibodies were used: antibodies against FXR (1:500, bs-12867R, Bioss), TGR5 (1:500, NBP2-23669SS, Novus), SREBP1 (1:800, ab28481, Abcam), PPARA (1:1000, 66836-1-Ig, Proteintech), phosphor-PPARA (P-PPARA, S12) (1:800, ab3484, Abcam), and GAPDH (1:1,000, 2118S, Cell Signaling Technology).
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