Ptp1b antibodies

N-Methyl-4-piperidone, Benzaldehyde, Diisopropylamine, n-Butyllithium (2.5 M solution in hexanes), 4-(Dimethylamino)pyridine (DMAP), and Sodium bicarbonate were purchased from Merck. Acetic acid, Dichloromethane, Tetrahydrofuran, Ethanol, Ethyl acetate, Citric acid, Sodium Sulfate(VI), and Acetic anhydride were purchased from POCH, Gliwice, Poland. Vanilin was purchased from Acros, Waltham, Massachusetts, USA. Dimethyl-sulfoxide (DMSO), Potassium superoxide, 18-crown-6-ether, Nitro blue tetrazolium chloride (NBT), Curcumin, Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), recombinant PTP1B phosphatase, para-nitrophenyl phosphate (pNPP), and 2′,7′-dichlorofluorescein were purchased from Sigma-Aldrich, Saint Louis, Missouri, USA. Dulbecco’s Modified Eagle’s Medium (DMEM) and Phosphate-buffered saline (PBS) were purchased from PAN-biotech, Aidenbach, Germany. Lastly, 4–20% MP TGX Tain-Free Gel 10W was purchased from Bio-Rad Laboratories, Hercules, CA, USA and PTP1B antibodies were purchased from Cell Signaling, Danvers, MA, USA.

The level of PTP1B phosphatase was determined by the Western blot technique. Briefly, the MCF-7 and MDA-MB-231 cell lines were non-treated (control) or treated with curcumin, compound 5, and compound 6 in concentrations of 25 µM, for 24 h, then the cells were harvested, centrifuged, and lysed. The protein concentration was determined using the Bradford reagent [56 (link)]. The proteins were separated on a 4–20% gradient of polyacrylamide gel by electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred to a methanol-activated PVDF membrane in the TBE buffer using a semi-dry transfer device (GE Healthcare, Chicago, IL, USA). Membranes were incubated with primary PTP1B antibodies (Cell Signaling, Danvers, MA, USA), and overnight, with horseradish peroxidase (HRP)-conjugated secondary antibodies. Visualization was performed using chemiluminescence enhanced with a chemiluminescence reagent according to the manufacturer’s protocol. The signal was read using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA). Protein levels were quantified using densitometry analysis by the ImageJ program [57 ]. The results were normalized to β-actin.