Total proteins were extracted from transfected cells using RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentration of the lysate was quantified using a bicinchoninic acid protein assay (BCA). Equal amounts of proteins were separated on a 7–10% SDS-PAGE gel and subsequently transferred to a methanol-activated NC membrane (Whatman, Dassel, Germany). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature and then incubated overnight with the following antibodies: LEF1 (ab53293, Abcam, 1/5000 dilution), E-cadherin (Cell Signaling Technology, 1/1000 dilution), vimentin (GTX100619, GeneTex, 1/5000 dilution), Snail (GTX100754, GeneTex, 1/500 dilution) and GAPDH (ab181602, Abcam, 1/10,000 dilution). GAPDH was used as a loading control. Then, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies (Biosynthesis Biotech, Beijing, China). Next, the membranes were imaged using an ECL Kit (Pierce, Shanghai, China) following the manufacturer’s instructions.
Gtx100754
Manufactured by GeneTex
GTX100754 is a compact, high-performance microcentrifuge designed for a wide range of laboratory applications. It features a brushless motor and digital speed control, allowing precise speed adjustment from 6,000 to 15,000 rpm. With a maximum RCF of 20,800 x g, this centrifuge is capable of efficiently separating a variety of samples, including cells, proteins, and nucleic acids. The unit comes equipped with a 24-place rotor and supports various tube sizes up to 2.0 mL.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (1 mentions)
Nc membrane, by Cytiva (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ab53293, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Gtx100619, by GeneTex (1 mentions)
Ripa lysis buffer, by Roche (1 mentions)
Whatman, by Cytiva (1 mentions)
Ab9829, by Abcam (1 mentions)
Ab137369, by Abcam (1 mentions)
Azd5363 s8019, by Selleck Chemicals (1 mentions)
D4540, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Gtx125891, by GeneTex (1 mentions)
Ab8984, by Abcam (1 mentions)
Ab16048, by Abcam (1 mentions)
3 protocols using gtx100754
1
Western Blot Analysis of EMT Markers
2
Antibody-Based Targeting of CCL20/MIP-3α Pathway
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Recombinant human (rh) CCL20/macrophage inflammatory protein 3 (MIP‐3) alpha antibody (MAB360‐100) was purchased from R&D Systems. The primary antibodies used in neutralizing, western blot (WB), and immunohistochemistry (IHC) assays were as follows: for WB, rabbit anti‐MIP‐3 alpha antibody (ab9829; Abcam), rabbit anti‐CCR6 antibody‐N‐terminal (ab137369; Abcam), rabbit anti‐AR antibody (ab133273), and rabbit anti‐Snail (3879 S; Cell Signaling Technology); for IHC, rabbit Snail antibody (GTX100754; Gene Tex); and rabbit phospho‐AKT (pAKT) antibody (Ser473; 9271 S; Cell Signaling Technology), rabbit AKT antibody (9272 S; Cell Signaling Technology), mouse anti‐GAPDH antibody (60004‐1‐Ig; ProteinTech), mouse Ki‐67 antibody (M7248; Agilent Technologies), and rabbit endomucin antibody (14–5851‐82; Thermo Fisher Scientific). HRP‐conjugated anti‐rabbit IgG antibody (7074 S) and HRP‐conjugated anti‐mouse IgG antibody (01803–44) were purchased from Cell Signaling Technology and Nacalai Tesque, respectively.
We purchased the AKT inhibitor AZD5363 (S8019) from Selleck Chemicals and the CCR6 inhibitor 1 (HY‐112701) from Med Chem Express. Both drugs were solubilized in dimethyl sulfoxide (D4540; Sigma‐Aldrich).
We purchased the AKT inhibitor AZD5363 (S8019) from Selleck Chemicals and the CCR6 inhibitor 1 (HY‐112701) from Med Chem Express. Both drugs were solubilized in dimethyl sulfoxide (D4540; Sigma‐Aldrich).
3
Immunofluorescence Imaging of Nuclear Lamins
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The primary antibodies used in this experiment are 1:200 dilution of anti-lamin A/C mouse monoclonal antibody (ab8984, Abcam), 1:1000 dilution of anti-lamin B1 rabbit polyclonal antibody (ab16048, Abcam), 1:200 dilution of anti-SNAI1 (GTX100754, GeneTex), and 1:200 dilution of anti-paxillin rabbit polyclonal antibody (GTX125891, GeneTex). The cell nuclei and F-actin were stained with 5 μg/mLof Hoechst 33342 (H3570, Thermo Fisher Scientific) and 165 nM of rhodamine–phalloidin (R415, Thermo Fisher Scientific), respectively. Immunofluorescent images were taken using a laser scanning microscope system (LSM 880, ZEISS) equipped with a 40× oil-immersion objective (plan-apochromat, ZEISS; numerical aperture = 1.3).
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