The total cellular proteins were extracted with the cell lysis buffer (P0013, Beyotime, Beijing, China) containing PMSF. The cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The total protein concentrations of the supernatants were estimated using the BCA kit (P0012S, Beyotime). Then, equal amounts of samples (30 μg) were separated on a 10% PAGE. The separated proteins were transferred onto the PVDF membranes (Merck Millipore). The membranes were blocked with 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4℃ with primary antibodies against MENA (ab244423, Abcam, 1:500), E-Cadherin (#3195, CST, 1:1000), Vimentin (sc6260, santa cruz, 1:1000), p-AKT (#4060, CST, 1:1000), AKT (ARG56418, Arigo, 1:1000), and β-actin (ab8227, Abcam, 1:1000). Then, they were incubated with the secondary antibodies at 37 °C for 120 min. The blots were developed using the ECL solution (P1050, Applygen, Beijing, China), and the protein bands were detected and quantified.
Arg56418
Manufactured by Arigo Biolaboratories
The ARG56418 is a high-precision laboratory centrifuge capable of separating a variety of liquid samples at high speeds. It features a durable, corrosion-resistant housing and a balanced rotor design for efficient and reliable operation.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (2 mentions)
Sc 6260, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
P0013, by Beyotime (1 mentions)
Ecl solution, by Applygen (1 mentions)
Ab183666, by Abcam (1 mentions)
Ab13370, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
2 protocols using arg56418
1
Protein Expression Analysis in Cells
2
Western Blot Analysis of Cellular Signaling Proteins
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The cells were lysed using RIPA lysis buffer (P0013B; Beyotime Institute of Biotechnology), containing a protease and phosphatase inhibitor cocktail. Lysate protein concentrations were determined using a BCA kit (P0010S; Beyotime Institute of Biotechnology). The denatured protein samples were separated by 10% SDS-PAGE in electrophoresis buffer and transferred to a PVDF membrane. The membranes were blocked in 5% milk for 2 h, then incubated with primary antibodies overnight at 4°C. The antibodies were directed against Rap1A (rabbit polyclonal, ab197673, 1:1,000; Abcam), β1 integrin (rabbit polyclonal, ab183666, 1:3,000; Abcam), matrix metalloproteinase (MMP)9 (rabbit polyclonal, 10375-2-AP, 1:1,000; ProteinTech Group, Inc.), E-cadherin (rabbit monoclonal, 24E10, 1:1,000; Cell Signaling Technology, Inc.), Slug (rabbit polyclonal, arg55242, 1:1,000; Abcam), AKT (rabbit polyclonal, arg56418, 1:600; Arigo), SP1 (rabbit polyclonal, ab13370, 1:3,000) and pAKTser473 (rabbit monoclonal, 4060, 1:2,000; Cell Signaling Technology, Inc.). The membranes were incubated with HRP-conjugated secondary antibodies for 2 h, and then antigen-antibody complexes were detected using an electrochemiluminescence kit to compare the corresponding protein expression levels among different samples.
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