Cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 1x EDTA-free protease inhibitors) and protein concentration was determined by the bicinchoninic acid assay method (BCA, Pierce) according to the manufacturer protocol. Samples were boiled for 5 min after the addition of 6 ×x Laemmli buffer (9% SDS and 60% glycerol, 375 mM Tris-HCl pH 6.8, 0.015% Bromophenol blue, 12% β-mercaptoethanol). Proteins were separated for 2 hr on a 12% SDS-polyacrylamide gel using a Bio-Rad mini-PROTEAN electrophoresis apparatus, transferred to low-fluorescence PVDF membranes (Millipore) for 2 hr at 4°C at a constant 70 V in 25 mM Tris, 150 mM glycine, and 10% (vol/vol) methanol transfer buffer, blocked with Odyssey Blocking Buffer (OBB, LI-COR Biosciences) for 1 hr at room temperature, and probed with 1:1000 dilution of mouse monoclonal anti-SCD antibody (ab19862; Abcam) in OBB overnight at 4°C. After washing with PBS-T, membranes were incubated with fluorescent goat anti-mouse secondary antibodies (926-32210; LI-COR Biosciences) at 1:15000 in OBB for 1 hr at room temperature. Signal was detected using an Odyssey Infrared Imaging System (LI-COR Biosciences). To ensure antibody specificity, shRNA knockdown of SCD1 was performed with two independent shRNA constructs (V3LHS_305870 and V3LHS_305872; Open Biosystems) and compared with non-silencing shRNA construct (RHS4346; Open Biosystems).
Ab19862
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab19862 is a primary antibody product from Abcam. It is a recombinant monoclonal antibody targeted against a specific protein. The core function of this product is to detect and bind to the target protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab174830, by Abcam (3 mentions)
Ab22759, by Abcam (2 mentions)
Ab81299, by Abcam (2 mentions)
Ab125066, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer obb, by LI COR (1 mentions)
Rhs4346, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab131215, by Abcam (1 mentions)
Ab6046, by Abcam (1 mentions)
Dhcr24, by Cell Signaling Technology (1 mentions)
Ab81298, by Abcam (1 mentions)
Sc 13119, by Santa Cruz Biotechnology (1 mentions)
T6793, by Merck Group (1 mentions)
Sc 965, by Santa Cruz Biotechnology (1 mentions)
Sc 13551, by Santa Cruz Biotechnology (1 mentions)
Sc 558, by Santa Cruz Biotechnology (1 mentions)
Hpa007415, by Merck Group (1 mentions)
T5168, by Merck Group (1 mentions)
Sc 15407, by Santa Cruz Biotechnology (1 mentions)
20 protocols using ab19862
1
Western Blot Analysis of SCD1 Protein
2
Western Blot Analysis of Lipid Metabolism Enzymes
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Western blot analysis was carried out as previously described (Corno et al., 2017 (link)). Lysates were fractionated by SDS-PAGE and proteins were blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk and then incubated overnight at 4°C with antibodies to FASN (HPA006461, Sigma-Aldrich, St. Louis, MO, United States), DHCR24 (2033, Cell Signaling Technology, Danvers, MA, United States), stearoyl–CoA desaturase 1 (SCD1) (ab19862 Abcam, Cambridge, UK), mevalonate kinase (MVK) (ab154515, Abcam), acetyl-CoA acetyltransferase 1 (ACAT1) (ab168342, Abcam), ACAT2 (ab131215, Abcam), glutathione peroxidase 4 (GPX4) (sc166120, Santa Cruz Biotechnology, Dallas, Texas, United States), β-Hydroxy β-methylglutaryl-CoA reductase (HMGCR) (ab174830, Abcam). An anti-β-tubulin antibody (ab6046, Abcam) was used as control for loading. Antibody binding to blots was detected by chemo-luminescence (Amersham Biosciences, Cologno Monzese, Italy). Secondary antibodies were obtained from GE Healthcare.
3
Antibody Validation for Western Blot and Immunofluorescence
4
Immunohistochemical Analysis of SCD1 Expression
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Immunohistochemistry was performed on tissue microarray (TMA) according to the standard streptavidin-peroxidase method (Zymed, San Francisco, CA, USA). Anti-SCD1 antibody (dilution 1:300; Abcam (ab19862), Cambridge, MA, USA) was used for immunohistochemistry staining. Meanwhile, antibody diluent (Abcam, ab64211) without primary antibody served as the control antibody. Results were recorded with Nikon eclipse Ti Microscope (Nikon Corporation, Tokyo, Japan) and Leica DM6000 B (Leica Microsystems, Wetzlar, Germany). The immunohistochemistry results were analyzed by two independent uropathologists who did not know the clinical condition of the patients. The expression level of SCD1 was scored by combining both the percentage of positive stained tumor cells and the staining intensity. As shown in Fig 1 , the intensity of staining was scored as: 0 (negative), 1 (weak positive), 2 (moderate positive), or 3 (strong positive), and scores representing the percentage of positive cells was graded as: 1 (0–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%). Comprehensive score = staining percentage × intensity. SCD1 expression level was classified as follows: <6, low expression; and ≥6, high expression.
5
Comprehensive Histological Analysis of Liver and Adipose Tissue
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Tissues were fixed with 10% (v/v) neutral buffered formalin. One part of each liver was embedded in a paraffin block, and the other part was frozen with Tissue-Tek O.C.T. Compound (Sakura) after cryoprotection with 30% sucrose. Paraffin-embedded livers were sliced into 5-μm sections and stained with hematoxylin and eosin (H&E) for immunostaining in this study. Frozen liver sections were cut into 5-μm sections and stained with Oil red O (Sigma) in 60% isopropanol or 0.25 mg/ml filipin. Paraffin-embedded inguinal white adipose tissue was sliced into 5-μm sections and stained with H&E or CellMask Orange Plasma membrane Stain (Thermo Fisher) with DAPI for nuclear staining. Adipocyte area and adipocyte counting were measured using ImageJ software in 6–8 different fields and are presented in a bar graph. For immunostaining, sections were incubated with the following primary antibodies: ACOT12 (MBS273137, Mybiosource), FASN (GTX109833, GeneTex), SCD1 (ab19862, Abcam), PPARα (ab8934, Abcam), and HA-tag (3724, Cell Signaling). Positive staining was visualized by detection with DAB Peroxidase (HRP) Substrate (SK-4105, Vector Laboratories), and counterstaining with hematoxylin. Histological images were acquired with a light microscope (Thermo Fisher).
6
Lipid Metabolism Regulation in Renal Cancer
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The 786-O, ACHN and HK2 were purchased from the Chinese Academy of Sciences cell bank. All cells were cultured in the presence of penicillin/streptomycin at 37 °C in air containing 5% CO2. The antibodies used included mouse anti-ABCA1 (Abcam, ab18180), rabbit anti-LDLR (Abcam, ab30532), rabbit anti-HMGCR (Abcam, ab174830), rabbit anti-SREBP-1c (Abcam, ab28481), mouse anti-SCD1 (Abcam, ab19862), rabbit anti-SRB1 (Abcam, ab217318), rabbit anti-FASN (CST, 3180), rabbit anti-Bax (Abcam, ab32503), rabbit anti-Bcl2 (Abcam, ab59348), rabbit anti-Caspase3 (Abcam, ab32042), and anti-HMGCR (used for IHC, Proteintech, 13533). Cholesterol-methyl-β-cyclodextrin (Sigma, C4951), SR9243 (MCE, 16972), LXR623 (MCE 10629), stearic acid (MCE, B2219), oleic acid (MCE, N1446) and palmitic acid (MCE, N0830) were also utilised.
7
Western Blot Analysis of Hepatic Lipid Metabolism
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Mouse liver tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors (Bimake, Houston, USA). The protein concentration was determined using a BCA Protein Quantitative Assay Kit (Shenergy Biocolor Bioscience & Technology Co., Shanghai, China). Total protein was mixed with SDS loading buffer and subjected to SDS-PAGE on a 10% gel. Proteins were electrotransferred onto PVDF membranes (Millipore) and the blots were probed with the following primary antibodies overnight at 4 °C: anti-FASN (cst3180, Cell Signaling Technology, USA), anti-SREBP1C (ab3259, Abcam, USA), anti-SCD1 (ab19862, Abcam), anti-CPT1α (ab176320, Abcam), anti-MTP (sc-135994, Santa Cruz Biotechnology, USA), anti-CD36 (18836-i-ap, Proteintech, China), anti-FGF21 (ab171941, Abcam), anti-BIP (11587-1-ap, Proteintech), anti-p-IRE (ab48187, Abcam), anti-IRE (ab37073, Abcam), anti-eIF2α (cst9722, Cell Signaling Technology), anti-p-eIF2α (cst3597, Cell Signaling Technology), anti-ATF4 (10835-1-ap, Proteintech), anti-ATF6 (ab122897, Abcam), anti-p-PERK (sc-32577, Santa Cruz Biotechnology), anti-PERK (ab65142, Abcam) and anti-GAPDH (60,004–1, Proteintech). Appropriate secondary antibodies conjugated to horseradish peroxidase (Amersham) were diluted 1:5000 used. The bound primary antibodies were visualized using the Alpha Q detection system.
8
Immunohistochemical Analysis of GC Markers
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Immunohistochemical staining was performed on 93 cases of GC and their paired adjacent tissues according to the standard method described previously using the following antibodies: anti-SCD1 (ab19862, 1:100, Abcam, Cambridge, UK), anti-YAP1 (14074S, 1:200, Cell Signaling Technology (CST), Danvers, MA, USA), anti-E-cadherin (14472S, 1:100, CST), anti-vimentin (5741S, 1:20, CST), anti-N-cadherin (13116S, 1:100, CST) and anti-TEA domain transcription factor 1 (TEAD1) (ab133533, 1:100, Abcam). Immunohistochemical (IHC) staining scores were evaluated based on the ratio and intensity of stained cells following the methods described in the previous study.12 (link) A categorisation of expression into three groups was based on the scores: negative, moderate and positive expression
9
Organoid Immunofluorescence Staining Protocol
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Organoids were dissociated from Matrigel using cell recovery solution (Corning) as aforementioned. Organoids were then processed as per published protocol57 (link). Organoids were stained with the following antibody: ADAR1 (1:100, Cell Signaling Technology, 14175), SCD1 (1:100, abcam, ab19862), and KHDRBS1 (1:1000, abcam, ab86239). Organoids were mounted and counterstained with antifade DAPI (Invitrogen).
10
Immunoblotting Lipogenic Enzyme Analysis
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Immunoblotting for the lipogenic enzymes, FASN and SCD-1, was performed as described previously [10 (link)]. In brief, V-BAP treated and control sub-confluent (80%) cultures of SAOS2, MG63 cells and human MSCs were harvested after 48 h by trypsinisation and pelleted by centrifugation. Following suspension in phosphate buffered saline (PBS) and re-pelleting, cell extracts were prepared by lysis using radioimmunoprecipitation assay (RIPA) buffer. Thirty micrograms of protein for each cell sample was combined with Laemmli 4× loading dye (Bio-Rad, Fisher Scientific Ltd.) and 10% β-mercaptoethanol (Sigma-Aldrich Ltd.) and heated for 5 min at 70°C. Cell extracts were separated using SDS-PAGE precast gradient gels (4%-15%; Bio-Rad), proteins transferred to Immobilon-P membrane (Millicorp, Sigma-Aldrich Ltd.). Membranes were sectioned in two parts at ∼60 KDa, the lower half probed with 1/1000 dilutions of anti-SCD-1 (ab19862; Abcam Ltd.), and the upper half with anti-ACC1 (4190; New England Biolabs Ltd.), anti-phospho-ACC Ser79 (11818; New England Biolabs Ltd.), and anti-FASN (ab22759; Abcam Ltd). The lower half of the membrane was probed with anti-β-actin antibodies (SigmaAldrich Ltd) diluted at 1/25,000 as loading control. Densitometry was performed using ImageJ software (http://rsb.info.nih.gov/ij/ ) and protein expression normalized to β-actin.
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