A549 and H1299 cells were inoculated in 6-well plates (6×105/well) respectively, and got treated according to grouping. After 72 hours of culture, total protein was extracted, and the BCA method was adopted to assess the protein concentration. Then, proteins were isolated on 10% polyacrylamide gel electrophoresis and transferred to the membranes. Afterwards, the membranes were blocked with skimmed milk and incubated with primary antibodies of HMGB1 (ab3047, 1:1000, Abcam), Vimentin (ab3047, 1:1000, Abcam), N-cadherin (ab3047, 1:1000, Abcam), E-cadherin (ab22759, 1:1000, Abcam) and GAPDH (ab22759, 1:1000, Abcam) at 4°C overnight. Subsequently, the membranes were incubated with goat anti-rabbit secondary antibody at room temperature for 2 hours. Finally, ECL chemiluminescence reagent was adopted for color rendering, gel imaging system was employed for imaging, and ImageJ software was conducted for grayscale analysis, with GAPDH as the internal reference.
Ab22759
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab22759 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab92726, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab28481, by Abcam (3 mentions)
Ab652, by Abcam (2 mentions)
Bca kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Ab133625, by Abcam (2 mentions)
Ab14041, by Abcam (2 mentions)
Ab68418, by Abcam (2 mentions)
Ab22595, by Abcam (2 mentions)
Itgb1, by Cell Signaling Technology (2 mentions)
Sc 376638, by Santa Cruz Biotechnology (2 mentions)
Thbs1, by Cell Signaling Technology (2 mentions)
Sparc, by Cell Signaling Technology (2 mentions)
Ab3269p, by Merck Group (2 mentions)
Ab19862, by Abcam (2 mentions)
Ab45174, by Abcam (2 mentions)
Ab3047, by Abcam (1 mentions)
Gb11130, by Wuhan Servicebio Technology (1 mentions)
Protease inhibitor cocktail, by Wuhan Servicebio Technology (1 mentions)
19 protocols using ab22759
1
Western Blot Analysis of EMT Markers
2
Quantifying Protein Expression Levels
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The GLUT1, FASN, and matrix metalloproteinase-2 (MMP2) expression levels were measured using Western blot analysis. Samples were lysed with a lysis buffer containing a protease inhibitor cocktail (G2006, Servicebio), and the protein concentrations were determined using a protein assay kit (G2006, Servicebio). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 20 μg of protein in each sample using 4% to 15% Mini Protean TGX precast gels (Servicebio). The following primary antibodies were used: anti-GLUT1 (AB652, 1:1000, Abcam), anti-FASN (AB22759, 1:1000, Abcam), and anti-MMP2 (GB11130, 1:1000, Servicebio). The bands were detected using a Western blotting detection system (Epson, Japan) and band intensity was calculated via densitometry using AlphaEaseFC (Alpha Innotech, USA).
3
Immunohistochemical Analysis of FASN and Ki67 in Tumor Sections
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Following paraffin embedding and sectioning (4 μM), the tumor tissue sections were dewaxed with xylene, hydrated with gradient alcohol, and treated with 3% H2O2 for 10 minutes. Microwave repair was made by applying 0.01 mol/L sodium citrate buffer (pH=6.0, 15 min), and the sections were blocked with 5% bovine serum albumin (BSA) for 20 minutes, followed by the incubation with anti-FASN (ab22759, Abcam, MA, USA) and Ki67 (ab15580, Abcam, MA, USA) overnight at 4°C. The following day, the goat-anti-rabbit IgG was added and kept at 37 °C for 60 minutes. After rinsing with PBS, DAB was used for color development. After hematoxylin re-staining, the sections underwent dehydration, permeation, sealing, and examination under a microscope (Olympus, Japan).
4
Protein Extraction and Western Blot Analysis
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Protein was extracted by Lysis buffer (KGP2100, KeyGEN BioTECH) and the concentration of protein was measured by bicinchoninic acid protein assay kit (KGSK3051, KeyGEN BioTECH). Fifty micrograms of protein was resuspended in sample loading buffer, boiled for 5 min, and electrophoresed on polyacrylamide gel. The proteins were transferred electrophoretically to a nitrocellu-lose membrane, blocked with PBS-Tween (0.05%) in 5% low-fat dry milk solution at room temperature for 2 h, and incubated with specific antibodies FASN (1:5000, 273kDa, ab22759, abcam), L-FABP (1:1000, 15kDa; ab7807, abcam), VEGF ( 1:500, 45kDa; bs-0279R, Bioss), VEGFR-2 ( 1:500, 151kDa; bs-0565R, Bioss), MEK5 (1:500, 49kDa; bs-4124R, Bioss), E-cadherin (1:500, 80kDa; bs-1016R, Bioss), or vimentin (1:500, 51 kDa; bs-0756R, Bioss) overnight at 4°C. The horseradish peroxidase-labeled goat-anti-rabbit (ZB 2301) or goat-anti-mouse (ZSGB-BIO) secondary antibodies were incubated at room temperature for 2 h. ECL (KGP1121, KeyGEN BioTECH) was used for chemiluminescence color reaction.
5
Immunohistochemical Analysis of Prostate Cancer
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For the immunohistochemical analysis of Cav-1/ACC1/FASN pathway we used tissue sections from 12 benign normal prostate epithelia, 15 untreated and 15 ADT treated primary PCa and 8 PCa bone metastases. All the samples were from patients enrolled in a clinical trial conducted in MD Anderson Cancer Center. Specimens were processed as previously described [10 (link)], and antibodies against Cav-1 (sc-894, Santa Cruz), ACC1 (#3662, Cell Signaling), and FASN (Ab22759, ABCAM) were used to stain for Cav-1, ACC1, and FASN, respectively. For the evaluation of cancer incidence and AR, PCNA, Cav-1, ACC1, and cleaved caspase-3 staining in mouseVPs, mouse tissues were processed as previously described [21 (link)]; the following antibodies were used: Cav-1 (sc-894) and AR (sc-816) from Santa Cruz, ACC1 (#3662) and cleaved caspase 3 (#9661) from Cell Signaling, and PCNA (AV03018) from Sigma Aldrich.
6
Quantitative Protein Analysis of sEVs
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sEV and cells were resuspended in RIPA lysis buffer (Millipore-Sigma) and incubated on ice for 20 min prior to centrifugation at 12,000 g for 10 min. Supernatants were used for western blotting in SDS-PAGE electrophoresis. Protein concentration was determined by a BCA kit (Thermofisher). The following antibodies were used: ADIPOQ (AB3269P, Millipore-Sigma), CD36 (ab133625, Abcam), HSP90alpha (PA3–013, Thermofisher), POSTN (ab14041, Abcam), PGAM1/4 (sc-376638, Santa Cruz); THBS1 (14778, Cell Signaling), ITGB1 (4706, Cell Signaling), EGFR (2232, Cell Signaling), SPARC (8725, Cell Signaling), IGFBP5 (AF578, R&D systems), CD9 (ab92726, Abcam), CD63 (ab68418, Abcam), CANX (ab22595, Abcam) and FASN (ab22759, Abcam). Given the large difference in the abundance for some of the proteins among different cell types, some of the blots were overexposed to show the signal in as many cell types as possible. Because of this, in some cases, the signal is beyond the linear range, and thus the quantification might not be absolutely accurate. Band intensity quantification was performed using ImageJ software (NIH).
7
Comprehensive Protein Expression Analysis
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Antibodies against CD63 (ab125011, 1:1000), α-SMA (ab32575, 1:100), FAP (ab207178, 1:1000), FSP (ab124805, 1:1000), GAPDH (ab8245, 1:10,000), CD9 (ab92726, 1:1000), CD81 (ab79559, 1:1000), FASN (ab22759, 1:500), ATP citrate lyase (EP704Y, 1:1000) and phospho-ATP citrate lyase (T447/S451) (ab53007, 1:1000) and USP2 (ab66556, 1:500) were purchased from Abcam (Cambridge, MA, USA). Antibodies against phosphor-PTEN (Ser380/Thr382/383) (9554S, 1:1000), total PTEN (7960 T, 1:1000), PDK1 (3062 T, 1:1000), phospho-PDK1 (Ser241) (3438 T, 1:1000), phospho-Akt (Ser473) (4060 T, 1:1000) and AKT (4691 T, 1:1000) were supplied by Cell Signaling Technology (Beverly, MA, USA). AKT inhibitor MK-2206 was provided by Selleck (Houston,USA).
8
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.
9
Immunohistochemistry Analysis of Cell Signaling Proteins
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IHC staining was used following the standard protocols. Paraffin-embedded tissue specimens were cut into 4-μm sections and deparaffinized. slides were incubated with anti-MEX3C (ab79041, Abcam, USA, 1:100 dilutions), FASN antibody (ab22759, Abcam, MA, USA, 1:100 dilutions), ACC1 antibody (21923–1-AP, Proteintech, Wu Han, China, 1:100) and SREBF1 (SERBP1) antibody (14088–1-AP, Proteintech, China, 1:100) overnight at 4°C, then incubated with a biotin-labeled secondary antibody (1:100 dilutions) for 40 mins at 37°C. Sections were incubated with 3,3′-diaminobenzidine (DAB), counterstained with hematoxylin, and visualized using light microscopy. Positive and negative controls were performed for each run of IHC. The staining signals were recorded by Olympus light, Tokyo, Japan microscope, and the staining index was measured and calculated by Olympus FV10-ASW software.
10
Quantitative Protein Analysis of sEVs
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