Protein extraction from NCI-H2030 and NCI-H1650 cells was implemented via the RIPA solution (P0013C; Beyotime, China). Protein concentrations were tested utilizing BCA assay kits (P0011; Beyotime). The extracted proteins were denatured in boiling water for 15-minutes. Thereafter, electrophoresis was carried out utilizing 10% SDS-PAGE gel for separation of protein molecules. Following protein transference onto PVDF membranes, the proteins were blocked in 5% nonfat milk at room temperature for 2 hours. Afterwards, incubation with primary antibody of β-catenin (1/1000; ab265591; Abcam, USA), E-cadherin (1/2000; ab231303), GAPDH (1/2500; ab9485), PKM2 (1/1000; #4053; Cell Signaling Technology, USA), phosphorylated PKM2 (p-PKM2; 1/1000; #3827; Cell Signaling Technology), lactate dehydrogenase A (LDHA; 1/1000; #3582; Cell Signaling Technology), and glucose transporter 1 (GLUT1; 1/1000; #73015; Cell Signaling Technology) was implemented at 4°C lasting 18 hours, followed by incubation with IgG-HRP goat anti-rabbit (1/3000; #7074; Cell Signaling Technology) for 2 hours. Signals were developed with ECL (Sigma-Aldrich) and normalized.
Ab265591
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab265591 is a primary antibody that targets an unspecified protein. It is designed for use in various laboratory applications. The product details and intended use are not available to provide an unbiased and factual description without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay kit, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
P pkm2, by Cell Signaling Technology (1 mentions)
Ripa solution, by Beyotime (1 mentions)
Ecl system, by Cytiva (1 mentions)
Ab264077, by Abcam (1 mentions)
Ab214821, by Abcam (1 mentions)
Ab40794, by Abcam (1 mentions)
Ab133612, by Abcam (1 mentions)
Ab81298, by Abcam (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab75777, by Abcam (1 mentions)
Enhanced chemiluminescent reagent, by Abcam (1 mentions)
A11753, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Radioimmunoprecipitation buffer, by Beyotime (1 mentions)
3 protocols using ab265591
1
Protein Extraction and Western Blot Analysis
2
Protein Expression and Signaling Pathways
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Protein (40 μg) was extracted using protein lysis buffer (Beyotime, Beijing, China), separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk, followed by incubation with primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Blots were developed using the ECL system (Amersham Biosciences, Little Chalfont, UK). The antibodies used in our study were purchased from Abcam (Cambridge, UK), including anti-β-catenin (phospho Y489) antibody (ab119801), anti-β-catenin antibody ab265591), anti-focal adhesion kinase (FAK) (phospho Y397) antibody (ab81298), anti-FAK antibody (ab40794), anti-bone morphogenetic protein (BMP2) antibody (ab214821), anti-osteocalcin (OCN) antibody (ab133612), anti-Runt-related transcription factor (RUNX2) antibody (ab264077). GAPDH was selected as internal reference gene. All experiments were repeated at least three times.
3
Osteoblast Protein Extraction and Analysis
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After two washes with PBS, the cells were treated with radioimmunoprecipitation buffer (Beyotime) containing 1 mmol L−1 phenylmethyl sulfonyl fluoride. Following a 1 minute ultrasonic shock, the MC3T3-E1 cells were centrifuged at 12 000g and stored at 4 °C for 10 minutes. The cell fragments were then discarded to obtain the total protein. The proteins were loaded onto a 7.5% SDS-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was incubated in 5% bovine serum albumin for 2 hours and then with primary antibodies, including ALP (1 : 1000, Abcam, ab154100), RUNX2 (1 : 1000, Abclonal, A11753), β-catenin (1 : 1000, Abcam, ab265591), phosphorylation of β-catenin (1 : 1000, Abcam, ab75777), and GAPDH (1 : 2500, Abcam, ab9485), which were incubated overnight at 4 °C. The membrane was visualized using an enhanced chemiluminescent reagent (Abcam), and GAPDH was used as an internal control. Secondary antibodies (1 : 5000, ab6721, Abcam) were applied for 2 hours in the analysis.
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