2.5. SDS-PAGE and Western-Blot

The total protein concentration was measured with a ULTROSPEC 2100 Pro UV-visible spectrophotometer (Biochrom US, Holliston, MA, USA) at the wavelength of 280 nm.

All proteins were separated by SDS-PAGE (Tris-HCl 7.5% gels). Cyst content was mixed with 2% β-mercaptoethanol containing 2x SDS loading buffer and pre-heated to 95 °C for 5 min. Each well was loaded with an equal protein amount: 20 µg of protein per well. SDS-PAGE gels were stained with a Coomassie brilliant blue R-250 stain.

Proteins were blotted to PVDF membrane (Bio-Rad, Hercules, CA, USA) and blocked with 5% skimmed milk solution. Separate membranes were probed with anti-human prolactin mAb (clone PRL02, dilution 1:1000, Invitrogen, Waltham, MA, USA) and anti-prolactin receptor mAb (clone B6.2, dilution 1:1000, Thermo Scientific, USA) primary antibodies, followed by goat-anti-mouse HRP-conjugated secondary antibodies (dilution 1:2000, Thermo Scientific, Waltham, MA, USA). Membranes were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). All captures were made with a Kodak Digital Image station 2000R (Eastman Kodak Company, Rochester, NY, USA). Each experiment was repeated in two replicates.