Antibodies to H3K27me3, JMJD3, p-Smad3, Smad3, p-AKT, AKT, p-ERK1/2, ERK1/2, Notch1, Jagged-1, UTX and DNMT1 were purchased from Cell Signaling Technology (MA, USA). Antibodies to fibronectin, α-SMA, collagen III, JMJD3, TGF-β1, Notch3 were purchased from Abcam (MA, USA). An antibody to Smad7 was obtained from Santa Cruz Biotechnology (CA, USA). An antibody to β-actin, PTEN and FBXW7 were obtained from proteintech (Wuhan, China). β-tubulin was purchased from Sigma-Aldrich (MO, USA). GSKJ4 was purchased from Selleck (Houston, USA). DMSO and other chemicals were obtained from Beyotime (Shanghai, China). Lipofectamine 3000 was obtained from Invitrogen-Thermo Fisher Scientific (CA, USA).
Fbxw7
Manufactured by Proteintech
Sourced in China
FBXW7 is a laboratory product that functions as an E3 ubiquitin ligase, which plays a role in the ubiquitin-proteasome system. It targets specific proteins for degradation by the 26S proteasome.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Cell Signaling Technology (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
α sma, by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Beyotime (1 mentions)
Jmjd3, by Cell Signaling Technology (1 mentions)
Jagged 1, by Cell Signaling Technology (1 mentions)
Antibodies to fibronectin, by Abcam (1 mentions)
Jmjd3, by Abcam (1 mentions)
Notch3, by Abcam (1 mentions)
Gsk j4, by Selleck Chemicals (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Tgf β1, by Abcam (1 mentions)
β tubulin, by Merck Group (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
Dnmt1, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
6 protocols using fbxw7
1
Antibody Validation for Signaling Pathways
2
Inhibition of c-Myc Activity
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Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich (MO, USA). The c-Myc inhibitor 10058-F4 was purchased from Beyotime (Shanghai, China). The ZC3H15 (cat. no. NBP1-81312) antibody was purchased from Novus Biologicals (Shanghai, China); The Tubulin (cat. no. 11224) and HA (cat. No. 51064) antibodies were purchased from Proteintech (Wuhan, China); The FBXW7 (cat. no. ab109617) and c-Myc (cat. no. ab32072) antibodies were purchased from Abcam (Shanghai, China); Flag (cat. no. 14793) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA).
3
Protein Expression Analysis by Western Blotting
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Cells were lysed using ice-cold RIPA lysis buffer (Beyotime) and the protein concentrations were quantified. Western blotting assay was performed as previously described (32 (link)). The PVDF membranes were incubated with rabbit anti-cyclin D1 (1:1,000; Proteintech, Wuhan, China), c-Myc (1:1,000; Cell Signaling, Boston, MA, USA), cyclin A2 (1:1,000; Cell Signaling), β-catenin (1:1,000; Cell Signaling), MMP2 (1:1,000; Proteintech), MMP7 (1:1,000; Cell Signaling), BTRC (1:1,000; Cell Signaling), FBXW7 (1:1,000; Proteintech), and α-tubulin (1:2,000; Proteintech) primary antibodies at 4°C for overnight. After conjugated with HRP goat anti-rabbit IgG antibody (1:5,000; Abcam, Cambridge, MA, USA), immunoreactive bands were visualized by the Super ECL prime (US Everbright Inc., Suzhou, China) under a western blotting detection instrument (Clinx Science Instruments, Shanghai, China).
4
Immunoblotting of Cellular Proteins
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Total protein was isolated from cells using lysis buffer (Beyotime, China). Immunoblotting was performed with primary antibodies against FBXW7 (Proteintech, China), MAP4 (Proteintech, China), MMP3 (Proteintech, China), VEGFA (Proteintech, China), ERK (CST, USA), phosphorylated ERK (Thr202/Tyr204) (CST, USA), GAPDH (Proteintech, China), His-tag (Proteintech, China), and GFP-tag (Proteintech, China). Secondary antibodies were purchased from CWbiotech (China). The signal was visualized using super enhanced chemiluminescence (ECL) detection reagent (Tanon, China).
5
Immunoblotting and FACS Analysis
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Immunoblotting was performed using the following antibodies diluted in TBS supplemented with 0.1% Tween 20 and 5% milk powder or 3% BSA: cyclin E1 (1:1000, Santa Cruz, sc-247), beta-actin (1:1000, Santa Cruz, sc-81178), p-p53 S15 (1:1000, Cell Signalling, 9284), p-CHK1 S345 (1:1000, Cell Signalling, 2348), p-RPA S4/S8 (1:5000, Bethyl Laboratories, A300-245), HA.11 (1:1000, BioLegend, 16B12), p53 (1:1000, Santa Cruz, sc-126), p21 (1:1000, Cell Signalling, 2947), HA (1:1000, Santa Cruz, sc-805), alpha-Tubulin (1:4000, Sigma, T5168), POT1 (1:1000, Novus Biologicals, NB500-176), FBXW7 (1:1000, Proteintech, 55290-1-AP), p-CDK1 T14/Y15 (in-house by Julian Gannon), Cdh1 (1:1000, in-house by Julian Gannon, AR38.2), anti-Mouse HRP (1:5000, Dako, P0447) and anti-Rabbit HRP (1:5000, Jackson Immuno, 711-035-152). The following antibodies were used for FACS and diluted in PBS supplemented with 1% BSA: MCM7 (1:200, Santa Cruz, sc-56324), cyclin B1 (1:200, Abcam, ab32053), p-H2A.X S139 (1:200, Millipore, 05-636), anti-Rabbit Alexa Fluor 555 (1:500, Thermo, A21428) and anti-Mouse Alexa Fluor 555 (1:500, Thermo, A21424).
6
Immunoblotting and Immunoprecipitation of Cell Death Regulators
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The following antibodies were used at the indicated dilution for immunoblotting (IB) and immunoprecipitation (IP) analyses: VDAC3 (1:800 for IB, 3 µg for IP; Proteintech, Wuhan, China), FBXW7 (1:800 for IB; 3 µg for IP, Proteintech), Ubiquitin (1:800 for IB, Proteintech), and β-Actin (1:8,000 for IB, Proteintech). Horseradish peroxidase (HRP) labeled secondary antibody conjugates were purchased from Molecular Probes (Abbkine, Beijing, China). Erastin (APExBIO, Beijing, China), ferrostatin-1 (Fer-1), rapamycin (Rapa), MK-2206, chloroquine (CQ), 3-MA, bafilomycin A1 (BafA1), MG132, and imidazole ketone Erastin (IKE) were obtained from Selleck Chemicals (Houston, TX).
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