Cells were counted, normalized for cell number, lysed in SDS sample buffer, separated by electrophoresis on NuPage 4–12% Bis-Tris gels (Novex) and blotted onto nitrocellulose membranes (GE Healthcare). Antibodies for PTBP1 (ab5642), Drosha (ab12286), DICR (ab14601), EXOSC6 (ab50910), EXOSC10 (ab50558), and PARN (ab188333) were obtained from Abcam. Antibodies for Upf1 (A300-036A), METTL3 (A301-567A), EXOSC4 (A303-775A), EXOSC5 (A303-887A), and Xrn1 (A300-443A) were obtained from Bethyl Labs. The antibody against ZAP (16820-1-AP) was obtained from Proteintech. The HIV-1 capsid antibody (183-H12-5C) was obtained from the NIH AIDS reagent repository. The GFP (G1546) antibody was obtained from Sigma. The HIV Env (12-6205-1) antibody was obtained from American Research Products. The HA (HA.11) antibody used in the CLIP assays was obtained from Biolegend.
Ab14601
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab14601 is a monoclonal antibody targeting the Ras-related C3 botulinum toxin substrate 1 (Rac1) protein. This product is suitable for use in various applications, including immunoblotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab12286, by Abcam (4 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (3 mentions)
Ab50558, by Abcam (2 mentions)
Ab5642, by Abcam (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ab50910, by Abcam (2 mentions)
Xrn1 a300 443a, by Fortis Life Sciences (2 mentions)
Antibody against zap 16820 1 ap, by Proteintech (2 mentions)
Gfp g1546 antibody, by Merck Group (2 mentions)
A1978, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab28943, by Abcam (2 mentions)
Ab57113, by Abcam (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab180947, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Gene pulser xcell electroporator, by Bio-Rad (1 mentions)
Electroporation cuvette, by Bio-Rad (1 mentions)
26 protocols using ab14601
1
Western Blot Protocol for Protein Detection
2
RNA-binding protein Immunoprecipitation Assay
3
Antibody-Loaded Microvesicle Electroporation
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DICER antibody was loaded onto MVs as previously described.17 Briefly, purified MVs were resuspended in electroporation buffer with a protein concentration equal to 0.3 µg/µl. We added 10 µg of DICER antibody (ab14601, Abcam) or 10 µg of rabbit IgG (ab172730, Abcam) into 500 µl of diluted MVs, and the mixture was loaded into electroporation cuvettes, with a gap width of 0.4 cm (Bio‐Rad). The electroporation was performed by the Gene Pulser Xcell™ Electroporator (Bio‐Rad) using the square wave protocol. Then, electroporated MVs were washed in PBS with 1% BSA. Pelleted MVs were resuspended in PBS again, and the same amount of MVs was assayed with the BCA kit before treating HUVECs.
4
Western Blot Protocol for DICER Analysis
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5
Comprehensive Western Blot Analysis Protocol
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Western blot analysis was performed as described previously40 (link). The following primary antibodies were used: anti-cyclin D1 (60186-lg, Proteintech, Chicago, IL, USA), anti-p27 (ab193379, Abcam, Cambridge, UK), anti-p21 (#2947, Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated Rb (Ser795) (#9301, Cell Signaling Technology), anti-IRS1 (#2382, Cell Signaling Technology), anti-phosphorylated IRS1 (Ser307) (D151214, BBI, Shanghai, China), anti-IGF1R (20254-1-AP, Proteintech), anti-phosphorylated AKT (Ser473) (66444-1-lg, Proteintech), anti-AKT (10176-2-AP, Proteintech), anti-phosphorylated mTOR (Ser2448) (D155324, BBI), anti-mTOR (20657-1-AP, Proteintech), anti-FoxP3 (D260367, BBI), anti-Dicer (ab14601, Abcam), anti-Drosha (55001-1-AP, Proteintech), and anti-GAPDH (#2118, Cell Signaling Technology).
6
Dicer Knockdown in HCT116 Cells
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HCT116 cells were transfected with shDicer1, shDicer2 or shCon plasmids, and grown under 1000 μg/ml Geneticin (G418) selection for 2–3 weeks. Knockdown of Dicer in G418-resistant monoclones was verified by western blot using anti-Dicer antibody (ab14601, Abcam, Cambridge, MA, USA).
7
Western Blotting for Argonaute and Dicer Proteins
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Argonaute protein levels were detected by western blotting. Briefly, the membrane was incubated in 5% milk in TBS-T blocking solution for 1 h at room temperature and in rabbit monoclonal primary (Ago1: CST#5053, Ago2: CST#2897) in 5% BSA, TBS-T with 1:1000 dilution for overnight at 4°C. The membrane was washed three times in 1× TBS-T and incubated with 1:5000 diluted HRP goat-anti-rabbit secondary in 5% BSA, TBST at room temperature. Immobilon Western Chemiluminescent HRP Substrate from Millipore was used for developing the signal.
Twenty micrograms of cell lysate from wild-type and Dicer knockout cells was loaded into 7.5% SDS-PAGE gel to detected Dicer protein levels by western blotting. Blocking was performed in 3% milk in PBS-T for 1 h at room temperature. 1:1000 dilution of Dicer antibody (Abcam ab14601) and 1:2000 dilution of α-tubulin antibody (Santa Cruz sc-5286) was used as a primary at 4°C for overnight.
Twenty micrograms of cell lysate from wild-type and Dicer knockout cells was loaded into 7.5% SDS-PAGE gel to detected Dicer protein levels by western blotting. Blocking was performed in 3% milk in PBS-T for 1 h at room temperature. 1:1000 dilution of Dicer antibody (Abcam ab14601) and 1:2000 dilution of α-tubulin antibody (Santa Cruz sc-5286) was used as a primary at 4°C for overnight.
8
Immunofluorescence Assay for DICER1
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Cells were planted in chamber 48 hrs prior to detection, and then fixed by incubating the slides in 10% formalin for 15 min. Cells were blocked in 2% goat serum (ab7481; Abcam, San Francisco, CA, USA); incubated with DICER1 (1:500; ab14601; Abcam, Burlingame, CA, USA) [Correction added on 21 May 2015 after first online publication: the value of DICER1 was added.] for at least 1 hr in PBST; incubated with Alexa Fluor® 488 (Goat antimouse IgG, Life Technologies, Bartlesville, OK, USA), for 30 min; washed in PBS, incubated for 10 min. with Hoechst (2 μg/ml, Life Technologies, Grand Island, NY, USA); and washed in PBS again. Fluorescence was visualized with a Leica (Leica Microsystems, Wetzlar, Germany) microscope (BD Biosciences).
9
RNA Immunoprecipitation of DICER in MDA-MB-231 Cells
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pcDNA3.1, pcDNA3.1-LINC01787, pcDNA3.1-LINC01787-mut, shCtl, shLINC01787-1, or shLINC01787-2 was transfected into MDA-MB-231 cells. Forty-eight hours after transfection, these cells were used to carry put RNA immunoprecipitation (RIP) assays with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) and a DICER specific antibody (5 μg per reaction; ab14601, Abcam, Cambridge, MA, USA) following the provided protocol.
10
Western Blot Analysis of RNAi Components
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Lysates from cells transfected with luciferase reporter plasmid DNA and siRNA were boiled in Laemmli Sample Buffer (Bio-Rad). Samples were separated on a 4–20% Criterion TGX Precast Gel (Bio-Rad). Protein was transferred to Whatman BA85 Nitrocellulose and protein was detected using the following antibodies: anti-Vimentin V9 (ab8069, Abcam, 1:10,000), anti-GAPDH 6C5 (ab8245, Abcam, 1:50,000), anti-Drosha (ab12286, Abcam, 1:1,000), anti-Dicer 13D6 (ab14601, Abcam, 1:1,000), Anti-Ago2 11A9 (SAB4200085, Sigma, 1:1,000). Primary antibodies were detected using the following horseradish peroxidase (HRP)-conjugated secondary antibodies: sheep anti-mouse IgG, HRP-linked Ab (NA931, GE Healthcare, 1:50,000), goat anti-rat IgG, HRP-linked Ab (NA935, GE Healthcare, 1:50,000), donkey anti-rabbit IgG, HRP-linked Ab (NA934, GE Healthcare, 1:50:000). Pierce ECL western blotting substrate (PI-32109) was used for detection.
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