Ab47742

Cells were seeded in 6-well plates; we incubated the cells at the time and zinc concentration indicated. Lysis was performed with 30 μL of lysis buffer containing 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, and EDTA-free protease inhibition cocktail (Roche). Lysates were vortexed for 30 min at 4 °C and centrifuged at 10,000× g to remove aggregates. Lysates were boiled for 5 min at 95 °C and placed on ice for 1 min. Then, 20 μL of each sample were loaded onto a 12% polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes using the iBlot system (Invitrogen, Waltham, MA, USA). Membranes were blocked with 5% milk in TBS-Tween 0.1% for 1 h at room temperature. Primary antibodies were diluted in blocking solution: anti-SERPINB2 (ab47742, Abcam) at 1:500, anti-Zip4 (20625-1-AP, Proteintech) at 1:500, and anti-GAPDH (ab8245, Abcam) at 1:1000 and incubated overnight at 4 °C. Anti-rabbit or anti-mouse HRP secondary antibodies (1:1000; GE Healthcare) were used. The ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA) was used to obtain high-quality images. Quantity One Software (Bio-Rad) was used to analyze the results.

Cells were grown in 10-cm dishes, exposed to treatments where required, and then lysed in 500 µl RIPA buffer with protease inhibitors (1.7 mg/ml aprotinin, 1 mg/ml leupeptin, 200 mmol/l phenylmethylsulfonyl fluoride, 200 mmol/l sodium orthovanadate, and 100 mmol/l sodium fluoride). Samples were then centrifuged at 13,000 rpm for 10 min and protein concentrations were determined using BCA protein assay according to the manufacturer’s instructions (Thermo Fisher Scientific, Milan, Italy). Equal amounts of whole-protein extract were resolved on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Merck Life Science, Milan, Italy), which were probed with primary antibodies against SERPINB2 (ab47742), GPER (ab137479) (Abcam, DBA, Milan, Italy), p-ERK1/2 (E-4), ERK2 (C-14) and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Bio-Rad, Milan, Italy) and then revealed using the chemiluminescent substrate for western blotting Clarity Western ECL Substrate (Bio-Rad, Milan, Italy). Densitometric analysis was performed using the freeware software ImageJ that allowed the quantification of the band intensity of the protein of interest with respect to the band intensity of the loading control.