Cells were lysed in RIPA buffer and proteins were separated on 10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking with 5% BSA-PBST buffer, the membranes were blotted by specific primary antibodies (1:1000 dilution) and HRP-conjugated secondary antibodies (1:5000 dilution). Finally, the signals on the membranes were visualized by ECL system (Thermo Fisher Scientific, Rochester, NY). We used the Bio-rad Image-lab 4.0.1 for collecting WB data. The MAO-A (sc-271123), GAPDH (sc-47724), tubulin (sc-23948), VEGF-A (sc-7269), AR (sc-816), p38 (sc-81621), and HIF-1α (sc-13515) antibodies were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The p-p38 antibody (#9211) was purchased from Cell signaling Technology (Danvers, MA). Rabbit HRP-conjugated second antibody (G21234 ) and mouse HRP-conjugated second antibody (G21040 ) were from Invitrogen (Carlsbad, CA).
Hif 1α sc 13515
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
HIF-1α (sc-13515) is a laboratory product offered by Santa Cruz Biotechnology. It is a protein that plays a key role in the cellular response to hypoxia, or low oxygen conditions. The product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
E cadherin 14472, by Cell Signaling Technology (1 mentions)
β actin sc 58673, by Santa Cruz Biotechnology (1 mentions)
Bay 87 2243, by Selleck Chemicals (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
β actin ab6276, by Abcam (1 mentions)
Ecl western blotting substrate kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cytiva (1 mentions)
Sc 8035, by Santa Cruz Biotechnology (1 mentions)
Hek 293 cell lines, by American Type Culture Collection (1 mentions)
C3h10t1 2, by American Type Culture Collection (1 mentions)
5 protocols using hif 1α sc 13515
1
Western Blot Analysis of Cellular Proteins
2
Melanoma and HEK293 Cell Culture
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WM35 and A375 melanoma cells and HEK293 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and authenticated and tested for contamination. They were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China) at 37°C in a humidified atmosphere containing 5% CO2.
BAY 87-2243 (S7309) were purchased from Selleckchem. HIF1α (sc-13515, 1:1000), β actin (sc-58673, 1:1000) and GAPDH (sc-32233, 1:1000) were obtained from Santa Cruz Biotechnology. The antibodies recognizing E-Cadherin (#14472, 1:1000) and Vimentin (#3390, 1:1000) were purchased from Cell Signaling Technology.
BAY 87-2243 (S7309) were purchased from Selleckchem. HIF1α (sc-13515, 1:1000), β actin (sc-58673, 1:1000) and GAPDH (sc-32233, 1:1000) were obtained from Santa Cruz Biotechnology. The antibodies recognizing E-Cadherin (#14472, 1:1000) and Vimentin (#3390, 1:1000) were purchased from Cell Signaling Technology.
3
Quantitative Protein Analysis by Western Blot
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HOXA9 (sc-81291, 1: 1000), HIF-1α (sc-13515, 1: 1000), and HK2 (sc-130358, 1: 1000) antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA). PDK1 (ab52893, 1: 1000), c-Myc (ab32027, 1: 1000), cyclinD1 (ab40754, 1: 1000), and β-actin (ab6276, 1: 10000) antibodies were purchased from Abcam (Cambridge, UK). Secondary HRP-conjugated antibodies against rabbit (#7074, 1: 100000) and mouse (#7076, 1: 100000) were products of Cell Signaling Technology (Beverly, MA). The lysates were prepared with RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. The concentration of protein lysates was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). We loaded 20 μg lysates on each well of the SDS-PAGE gel. The proteins were separated and transferred to a PVDF membrane. After that, membranes were blocked in 5% non-fat milk followed by incubation with primary antibody overnight at 4°C. On the next day, the membrane was incubated with secondary antibody for 1 h at room temperature. The blots were developed with an ECL Western Blotting Substrate Kit (Thermo Fisher Scientific).
4
Western Blot Analysis of HIF-1α and Lamin A/C
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Transfected NP cell lysates were boiled in an SDS sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. After the transfer, the membranes were blocked in 5% skim milk in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 40 min and incubated with the specific primary antibodies in the blocking solution for overnight at 4 °C. Antibodies against HIF-1α (sc-13515, 1:1000) and α-Tubulin (sc-8035, 1:1000) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Lamin A/C (#4777, 1:1000) was purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody followed by detection using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
5
Mesenchymal Cell Differentiation Assay
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C3H10T1/2, C2C12, 3T3L-1, and HEK-293 cell lines were purchased from ATCC. MEFs was extracted from embryo of NIH mouse. Lox speci c inhibitor, β-aminopropionitril (BAPN, #T13475) was purchased from Topscience Co., Ltd (Shanghai, China), and dimethyl sulfoxide was used as solvent. Cells were cultured with complete DMEM medium (containing 10% fetal bovine serum, 100 kU/L penicillin and 0.1 g/L streptomycin) at 37 ℃ with 5% CO 2 . Primary antibodies against GAPDH (10494-1-AP) and sclerostin (21933-1-AP) were ordered from proteintech (China branch); primary antibodies against BMP9 (sc-514211), LOX (sc-32409), Runx2 (sc-390351), OPN (sc-21742), β-catenin (sc-7963), H 2 A x (sc-517336) and HIF-1α (sc-13515) were all purchased from Santa Cruz Biotechnology (China branch).
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