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10 protocols using ab133485

1

Immunoblotting for Kinase Signaling

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Western blotting was followed, was described elsewhere [12 (link)]. Polyclonal rabbit antibody for Fli-1 (ab133485), PKCδ (ab182126), Phospho- PKCδ (ab133456), MEK (ab178876), Phospho-MEK (ab96379), BAD (ab32445), and Phospho-BAD (ab129192) were obtained from Abcam (Abcam, Cambridge, UK); ERK (#4695) and phospho-ERK (#9101) from Cell Signalling Technology (CST, Danvers, MA01923, USA), β-actin (20536–1-AP) and GAPDH (13937–1-AP), from Proto-Technology (Protein-Tech, Bucuresti, Romania). Antibody dilution according to the manufacturer instructions.
The inhibitor of MEK (U0126) [#S1102] were obtained from Sellectchem (Sellectchem, Houston, USA) and PKC agonist Phorbol 12-myristate 13-acetate (TPA) from Sigma, (Sigma, St. Louis, MO, USA). These compounds were dissolved in DMSO and added to the cells at indicated concentration, as described in the results section.
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2

Western Blot Analysis of Protein Targets

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Total protein from cell lines was extracted using RIPA buffer (Beyotime Institute of Biotechnology, CN) containing 1:100 PMSF (Solarbio, CN). The protein concentration was determined using a BCA kit (Solarbio, CN) according to the manufacturer's protocol. Load equal amounts of protein into the wells of the SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked using non-fat milk for 1 h at room temperature. Incubate the membrane with primary antibody in blocking buffer overnight at 4°C. After washed by TBST (Beyotime Institute of Biotechnology, CN) at room temperature for three times, the membrane was incubated with Anti-rabbit IgG (H + L) DyLight™ 800 4X PEG Conjugated secondary antibody (5151s, Cell Signaling Technology, US) in blocking buffer at room temperature for 1 h. The following primary antibodies were used: anti-FLI1 (ab133485, Abcam, UK), anti-UBASH3A (15,823–1-AP, Proteintech, DE), anti-UBASH3B (19,563–1-AP, Proteintech, DE), polyclonal rabbit test primary antibodies; anti-GAPDH (G9545, Sigma Aldrich, US). Antibody dilution was conducted according to the manufacturer’s instructions. The Odyssey system (LICOR Biosciences) was used for western blot membrane imaging and analysis.
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3

Protein Expression Profiling in A673/TR/shEF1 Cells

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Protein from A673/TR/shEF1 cells was extracted at d0, d7, d11, d14, and d17 with RIPA and anti-protease cocktail (Roche). Western blots were performed following routine protocols and specific band detection was achieved by the use of rabbit monoclonal anti-FLI1 antibody (1:1000, ab133485; Abcam)38 (link), rabbit polyclonal anti-MYBL2 antibody (1:500, sc-725; Santa Cruz)39 (link), and mouse monoclonal anti-ß-actin (1:10,000, A-5316; Sigma-Aldrich). Anti-rabbit IgG horseradish peroxidase-coupled antibody (1:3000, Amersham Bioscience) and anti-mouse IgG horseradish peroxidase coupled antibody (1:3,000; Amersham Bioscience) was used as secondary antibody. Proteins were visualized using chemiluminescence (Pierce ECL Western blot chemiluminescent substrate; Thermo Fisher Scientific).
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4

FLI1 Immunofluorescence in A673 Cells

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The A673/Cas9/sgRNA and A673/sgRNA (control) cells were seeded on glass cover slides. After 72 h, the growth medium was removed, and the cells were washed in PBS. Then, cells were fixed and permeabilized for 15 min in ice-cold methanol. After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum (v/v) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK). The next day, cells were washed 4 × 10 min with PBS and incubated for 1.5 h at room temperature with the secondary antibody (1:500; Goat anti-rabbit IgG-Alexa Fluor 594, Abcam, Cambridge, UK) supplemented with 4% FBS. Cells were counterstained for 4 min with the nuclear stain 4,6-diamidino-2-phenylindole (DAPI), washed 4 × 10 min with PBS, and mounted on slides using ProLong™ Gold antifade mounting medium (#P36934, Thermo Fisher Scientific, Waltham, MA, USA). Cells were visualized in a fluorescence microscope (Leica, Wetzlar, Germany).
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5

Whole-cell extract immunoblotting analysis

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Whole-cell extracts were prepared with protein lysis buffer (50mM Tris-HCl at pH 7.4, 1% Triton X-100, 0.1% SDS, 150mM NaCl, 1mM EDTA, and 1mM DTT), with addition of cocktail protease inhibitor tablets (Complete, Roche). Membranes were stained with FLI1 (ab133485; Abcam) (used to detect the EWSR1-FLI1 translocation), STAG2 (sc81852; Santa Cruz), p53 (sc126; Santa Cruz) or p16 (554079; BD Pharmingen) antibodies. ACTIN (sc1616; Santa Cruz) and VINCULIN (sc73614; Santa Cruz) antibodies were used as loading controls. Membranes were visualized with Odyssey CLx Imaging System (LI-COR Biosciences).
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6

Immunoblot Analysis of Hematopoietic Regulators

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Cells were collected and lysed in RIPA buffer with proteinase inhibitors. Immunoblot was performed using the standard protocol. Primary antibodies were used according to the manufacturer’s specifications: anti-CKIP-1 (D20, Santa Cruz), anti-GATA-1 (M20, Santa Cruz), anti-GATA-2 (ab109241, Abcam), anti-Fli-1(ab133485, Abcam), anti-c-Myb (ab109127, Abcam), anti-c-Myc (ab32072, Abcam), anti-NF-E2 (ab140598, Abcam) and anti-GAPDH (MBL).
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7

Western Blot Analysis of EMT Markers

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The expression of FLI‐1 (ab133485, Abcam), E‐cadherin (ab40772, Abcam), β‐catenin (#8480, CST), N‐cadherin (610920, BD bioscience), vimentin (550513, BD bioscience), ALDH1A1 (PA5‐32127, Thermo), and CD133 (MB0160, Bioworld) was determined by Western blotting. Briefly, the cells after transfection were lysed using the radioimmunoprecipitation assay (RIPA) buffer (Lot #71728155; MultiSciences, Hangzhou City, China) and 10 mmol/L phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 12 000 g at 4°C for 10 minutes to obtain the supernatant. The content of protein in the supernatant was measured using the BCA Kit (P0010; Beyotime, Nanjing, China). The protein samples (20‐50 μg/lane) were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with 10%‐12% acrylamide gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. The expression of protein was detected using the indicated antibodies, and then visualized by enhanced chemiluminescence (ECL) (Cat. no. 10200; NCM Biotech, Suzhou, China).
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8

Western Blot Analysis of Chromatin Proteins

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Cells were trypsinized, counted, washed with ice-cold PBS and lysed in Laemmli buffer (50 mM Tris-HCL, 2.5 mM EDTA, 2.55 mM EGTA, 2% SDS 20%, 5% Glycerol, 1% Bromophenol blue, protease inhibitor cocktail tablets and 2 mM DL-Dithiothreitol solution) at 10 million cells/ml. Protein lysates were sonicated and denatured at 95 C for 5 min and electrophorated on 4-15% Mini-PRO-TEANÒTGXTM gels (456-1084, BIO-RAD), transferred onto nitrocellulose membranes (1704159, BIO-RAD). Membranes were incubated overnight at 4 C with mouse anti-STAG2 (1:1,000, Santa Cruz Biotechnology, sc-81852), goat anti-STAG1 (1:5,000, ab4457, Abcam), rabbit anti-H3 (1:50,000, ab1791, Abcam), mouse anti-b-Actin (1:20,000, A5316, Sigma-Aldrich), rabbit anti-FLI1 antibody (1:1,000, ab133485, Abcam) and rabbit anti-H3K27ac (1:1,000, ab4729, Abcam). Then membranes were incubated 1h at room temperature with respective anti-rabbit, anti-mouse immunoglobulin G horseradish peroxidase (HRP) coupled secondary antibody (1:3,000, NA934 or NXA931, respectively; GE Healthcare) or anti-goat IgG-HRP (1: 10,000, SC-2354, Santa Cruz). Proteins were visualized using SuperSignalÔ West Pico Plus (34580, Thermo Scientific) and ChemiDocÔ Imaging System (BIO-RAD).
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9

Immunohistochemical Analysis of FLI1 and Ki67

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The sections were dewaxed in xylene and rehydrated using alcohol (gradients of 100%, 95%, 75%). The samples were then boiled in ethylenediaminetetraacetic acid solution for 20 min. The slides were incubated with primary antibodies to FLI1(1:1000, ab133485, Abcam) and Ki67 (1:500, #12202, Cell Signaling Technologies) at 37 C for 2 h and with the secondary goat anti-rabbit antibody IgG H&L (HRP) (1:2000, ab205718, Abcam) for 30 min. Subsequent color development was performed with Pierce™ DAB substrate kit (Thermo Fisher), and hematoxylin was used to counter-stain the nuclei. After dehydration, clearing and sealing, the slides were examined under a microscopy (Zeiss, Oberkochen, Germany). The proportion of stained cells and intensity of staining were scored by three pathologists who were unaware of the grouping. The percentage of positively stained cells was scored as <5% (0), 5-25% (1), 25%-50% (2), 50%-75% (3), and >75% (4). The intensity of staining was classified as no staining (0), light brown (1), brown (2), and dark brown (3). The final immunoreactivity score was the stained cell proportion  staining intensity (from a range 0 to 12).
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10

Western Blot Protein Quantification

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Protein extraction was conducted using ProteoPrep ® Total Protein Extraction Kit (Merck Millipore, Darmstadt, Germany). Quantification was performed using the BCA method (PA115, TIANGEN Biotech Co., Ltd., Beijing, China) as per the manufacturer's instructions. The extracted proteins (40 μg) were mixed with the loading buffer, denatured, separated, and blotted onto nitrocellulose membranes. The membranes were sealed with 6% skim milk and incubated with primary antibodies to DNMT3b (1:1000, 72,335, Cell Signaling Technologies, Beverly, MA), FLI1 (1:1000, ab133485, Abcam, Cambridge, UK) and GAPDH (1:3000, ab9485, Abcam, Cambridge, UK) overnight at 4 C. The membranes were then incubated with horseradish peroxidase-conjugated goat antirabbit secondary antibody (1:2000, ab205718, Abcam) at room temperature for 2 h. The membranes were washed and developed by Novex™ ECL chemiluminescent substrate kit (Thermo Fisher Scientific Inc., Waltham, MA) and quantified by quantity one.
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