20 μg protein extract were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk and followed by incubating with the primary antibody (FUT4, AP12067b, Abgent; cleaved caspase-3 ab2302, Abcam; cleaved PARP, ab4830, Abcam; Sp1, ab13370, Abcam; CD44, ab157107, Abcam; GSK-3β, 22,104–1-AP, Proteintech; p-GSK-3β, 22,104–1-AP, Proteintech; β-catenin, 51,067–2-AP, Proteintech; CyclinD1, 60,186–1-Ig, Proteintech; GAPDH, AP7873a, Abgent) on a shaker overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (rabbit IgG, 1/1000 diluted; UK). A GAPDH antibody (1/200 diluted; Santa Cruz Biotech) was used as a control. All bands were detected using ECL Western blot kit (Amersham Biosciences, UK). The bands were measured with LabWorks (TM ver4.6, UVP, BioImaging systems).
β catenin 51067 2 ap
Manufactured by Proteintech
Sourced in China, Japan
β-catenin (51067-2-AP) is a primary antibody that detects the β-catenin protein. β-catenin is a key mediator of the canonical Wnt signaling pathway and plays a role in cell-cell adhesion as a component of the cadherin protein complex.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl western blot kit, by Cytiva (1 mentions)
Cleaved caspase 3 ab2302, by Abcam (1 mentions)
Gsk3β 22104 1 ap, by Proteintech (1 mentions)
Cyclind1 60 186 1 ig, by Proteintech (1 mentions)
C myc ab32072, by Abcam (1 mentions)
Proliferating cell nuclear antigen (pcna), by ABclonal (1 mentions)
Lamina c, by ABclonal (1 mentions)
Ab134175, by Abcam (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Yeasen (1 mentions)
Ripa lysis buffer, by Yeasen (1 mentions)
Zb 2301, by ZSGB-BIO (1 mentions)
Bcl 2 12789 1 ap, by Proteintech (1 mentions)
Pi3k 20584 1 ap, by Proteintech (1 mentions)
Gapdh 10494 1 ap, by Proteintech (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Pakt 4060, by Cell Signaling Technology (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Azd5363, by Beyotime (1 mentions)
5 protocols using β catenin 51067 2 ap
1
Western Blot Analysis of Protein Markers
2
Comprehensive Protein Extraction and Analysis
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Total protein was extracted using RIPA lysis buffer from Yeasen (China). Cytoplasmic and nuclear protein extraction was carried out using the Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China). ACTIN and LaminA/C were used as internal controls for cytoplasmic and nuclear proteins, respectively. Primary antibodies used in this study were, c-Myc(ab32072, Abcam, USA), PCNA (A0264, ABclonal, China), SRSF1 (sc-33652, Santa Cruz Biotechnology, China), β-catenin (51067-2-AP, Proteintech, USA), cyclin D1(ab134175, Abcam, USA), LaminA/C (A0249, ABclonal, China), and ACTIN (AC026, ABclonal, China).
3
Fungal Compound SAF: Signaling Pathway
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SAF was isolated from a fungal strain in a lab from the Department of Pharmaceutical Sciences of Binzhou Medical University. SAF was dissolved in dimethyl sulfoxide (DMSO) and was stored at −20 °C as a stock solution. MARCH1 (bs-9335, Bioss, Beijing, China), phospho-AKT Ser473 (Sikh Association of Baltimore, Randallstown, MD, USA), total AKT (21054, SAB), GAPDH (10494-1-AP, Proteintech, Wuhan, China), PI3K (20584-1-AP, Proteintech, Tokyo, Japan), β-catenin (51067-2-AP, Proteintech), Mcl-1 (16225-1-AP, Proteintech), Bcl-2 (12789-1-AP, Proteintech), cleaved caspase-3 (9661, Commonwealth Soap & Toiletries, Fall River, MA, USA), cleaved caspase-7 (8438 CST), MG 132 (HY-13259, MedChem Express, Monmouth Junction, NJ, USA) and secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG; ZB-2301, ZSGB-BIO, Beijing, China) were obtained commercially.
4
Comprehensive Antibody and Inhibitor Protocol
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The LRP5 (#5731), p-Akt (#4060), pan-Akt (#4685), and E-cadherin (#14472) antibodies are all manufactured by Cell Signaling Technologies (Beverly, MA, USA). The p-mTOR (AP0115), mTOR (A2445), β-Actin (AC026), and PCNA (A12427) were purchased from Abclonal (China). The β-Catenin (51067-2-AP) and HRP-conjugated GAPDH (HRP-60004) are from Proteintech. The cleaved caspase-3 (ab32351) antibody is bought from Abcam (Cambridge, UK). The Akt inhibitors included GSK690693, AZD5363 and LY294002 and crystal violet are all manufactured by Beyotime (China). The alamarBlue is from thermofisher (Cleveland, OH, USA).
5
Investigating Sintilimab's Effects on TNBC Cells
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MDA-MB-231 and 4T1 cells were purchased from Beijing Union Medical College Hospital Cell Resource Sharing Platform, SUM159 cells were a gift from Dr. Jianjun He. MDA-MB-231 was cultured in L-15 medium, SUM159 was cultured in DMEM/F-12 medium, and 4T1 was cultured in RPMI 1640 medium. All cell lines were stored in a thermostat incubator at 37 °C, 5 % CO2.
MDA-MB-231 and SUM159 were prepared extracting proteins after adding sintilimab at a concentration gradient of 0.5, 1, 2, and 4 μg/mL following with incubating for 24 h, and then, western blotting assay were conducted. ALDH1A1 antibody was purchased from Abcam (ab52492), PD-L1 antibody was purchased from Cell Signaling Technology (#13684), β-catenin (51067-2-AP) and GAPDH (HRP- 60004) antibodies were from Proteintech. Whole cell lysates were prepared with RIPA buffer, proteins were separated by SDS/PAGE gel, transferred to PVDF membrane (Millipore), incubated with primary antibody overnight, followed by HRP-conjugated secondary antibody (Proteintech, SA00001-2) and detected chemiluminescent signals.
MDA-MB-231 and SUM159 were prepared extracting proteins after adding sintilimab at a concentration gradient of 0.5, 1, 2, and 4 μg/mL following with incubating for 24 h, and then, western blotting assay were conducted. ALDH1A1 antibody was purchased from Abcam (ab52492), PD-L1 antibody was purchased from Cell Signaling Technology (#13684), β-catenin (51067-2-AP) and GAPDH (HRP- 60004) antibodies were from Proteintech. Whole cell lysates were prepared with RIPA buffer, proteins were separated by SDS/PAGE gel, transferred to PVDF membrane (Millipore), incubated with primary antibody overnight, followed by HRP-conjugated secondary antibody (Proteintech, SA00001-2) and detected chemiluminescent signals.
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