LR were prepared using a previously described protocol with minor alterations [11 (link)]. Briefly, cells grown to confluence in 60 × 15 mm dishes and treated for 72 h with NB-DNJ at concentrations of 50 or 100 µM. All procedures were performed on ice or at 4 °C. Cells (2 × 106) were solubilised in 1% Lubrol in 50 mM Tris, 150 mM NaCl, pH 7.6 and 50 µL protease inhibitors. Homogenisation was performed by passing the cell lysate 20 times through a 21 G needle. The lysates were then centrifuged at 1500× g for 20 min at 4 °C and the supernatant loaded onto a discontinuous sucrose gradient as described before. Gradients were centrifuged at 4 °C for 18 h at 100,000× g using a Beckman SW40 rotor. Ten fractions were collected on ice from top to bottom, with fractions 1–3 containing predominately the LRs fractions, and 8–10 containing predominately the detergent-soluble cell fraction. The sucrose content was quantified from each gradient fraction by using a HI 96801 refractometer (HANNA instruments). The quality and location of LR was assessed by Western blotting of the gradient fractions using anti-FLOT2 antibody (1:5000; sc-28320 Santa Cruz, 200µg/mL, Santa Cruz, California USA).
Sc 28320
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-28320 is a lab equipment product from Santa Cruz Biotechnology. It is a device designed for scientific research applications. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Sw40 rotor, by Beckman Coulter (1 mentions)
Hi 96801 refractometer, by Hanna Instruments (1 mentions)
Anti ki67, by Proteintech (1 mentions)
Anti cd109, by Santa Cruz Biotechnology (1 mentions)
Anti flot 2, by Santa Cruz Biotechnology (1 mentions)
Dab staining kit, by ZSGB-BIO (1 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
Ar0024, by Boster Bio (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using sc 28320
1
Lipid Raft Isolation and Characterization
2
IHC and HE Staining of Paraffin Slides
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For IHC assays, the paraffin-embedded slides were deparaffinized, performed for antigen retrieval, and blocked with a proper blocking solution. The slides were then incubated with the primary antibody overnight at 4°C. The next day, the slides were incubated with relative secondary antibodies at 37°C for 30 min, and stained with a DAB staining kit (#ZLI9017, ZSGB BIO). At last, the slides were rehydrated. The following antibodies were used for studies: anti-FLOT2 (1:30, #SC-28320, Santa Cruz); anti-CD109 (1:30, #SC-271085, Santa Cruz); anti-Ki67 (1:2000, #SC-28320, Proteintech).
For HE assays, the paraffin-embedded slides were deparaffinized, stained with hematoxylin-eosin, and rehydrated.
For HE assays, the paraffin-embedded slides were deparaffinized, stained with hematoxylin-eosin, and rehydrated.
3
Immunofluorescence Analysis of NPC Cells
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NPC cells were seeded into the cover slide of 24-well plates. The next day, cells were fixed with 4% paraformaldehyde for 5 min. 5% FBS was used to incubate cells for 1-2 h at room temperature. The cells were incubated with primary antibodies overnight at 4°C. Relative fluorescently labeled secondary antibodies were used to incubate cells at room temperature for 2 h in the dark. 5 μg/ml of DAPI solution was used to stain the cell nuclear. Pictures were collected with a laser scanning confocal microscope (ZEISS). The following antibodies were used for studies: anti-FLOT2 (1:30, #SC-28320, Santa Cruz); anti-STAT3 (1:50, #10253-2-AP, Proteintech); anti-CD109 (1:100, #AF4385, R and D Systems).
4
Immunohistochemical Analysis of Flotillin-2
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Representative areas of tumor cores in the paraffin embedded tissue blocks were selected according to hematoxylin-eosin (H&E) staining, and then transferred into a recipient master. Antigen retrieval was performed by immersing the sections in citrate buffer (pH 6.0; cat. no. AR0024; Boster Biological Technology) and heating in a microwave for 5 min. The sections were subsequently treated with 3% hydrogen peroxide for 30 min at room temperature to quench endogenous peroxidase activity, followed by 10% normal goat serum for 30 min at 37°C. Sections were incubated with mouse monoclonal antibody against human flotillin-2 (cat. no. sc-28320; 1:200; Santa Cruz Biotechnology, Inc.) overnight at 4°C followed by incubation with anti-mouse secondary antibody for 30 min at 37°C. The sections were subsequently stained with DAB (Beyotime), and the staining was evaluated by two independent observers.
5
Lipid Raft Components Analysis
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Methyl-β-cyclodextrin (MβCD), soluble cholesterol, mouse anti-human actin monoclonal antibody (AC-40), anti-phosphotyrosine monoclonal antibody (PY20; P4110), inhibitor of Src-family tyrosine kinase (PP2), non-conjugated F (ab')2 fragment of goat anti-mouse IgG (M0659) and anti-tubulin monoclonal antibody (T4026) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-human β-adducin antibody (SC-376063) and antibody to flotillin-2 (SC-28320) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). AlexaFluor-488-conjugated cholera toxin (CTxB) was obtained from Molecular Probes (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Iscove's modified Dulbecco's medium was from Gibco (Thermo Fisher Scientific, Inc.). The β-adducin truncated mutant plasmid was produced in our laboratory.
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