Roti load 1

Sample preparation of snap-frozen tissue (human normal anterior pituitary gland and acromegalic pituitary tumors) was performed as follows: tissue samples were homogenized in ice-cold RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP–40, 0.5% sodium deoxycholate and 0.1% SDS) using an Ultra-Turrax. GH3 cell lysates were in RIPA buffer and disrupted using a 20G insulin needle. Protein concentration was determined with the Bradford assay. Immunoblot was performed using 10–15 µg to total protein in sample buffer (Roti-Load 1, Roth). Signals were detected using ECL Clarity (Biorad).

Co-immunoprecipitation experiments in GH3 cells were performed on the nuclear fractions. For fractionation cells were collected by careful scraping and pelleted by centrifugation. The cell pellet was carefully resuspended in hypotonic cell lysis buffer and incubated on ice for 15 min. Following centrifugation, the supernatant was decanted and cells were disrupted again in hypotonic lysis buffer using a 20G insulin syringe and centrifuged. The supernatant containing the cytoplasmic fraction was separated and 75 µl of cell extraction buffer was used to resuspend the pellet. A final centrifugation step was performed and the supernatant containing the nuclear fraction was separated to a clean tube and subjected to preclearing for 30 min at 4 °C using 10 µL Protein G Dynabeads per 10⁶ cells. The immunoprecipitation reaction was performed using Protein G Dynabeads coupled to the primary antibody in a separate reaction. In total, 10 µL of the antibody-bead complexes were given to 30 µL of precleared lysates. Following rotation overnight at 4 °C, immune complexes were washed and suspended in sample buffer (RotiLoad 1, Roth) to be immunoblotted as described above.

VLP samples were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). In total, 50 $\mathsf{\mu }$ L sample was mixed with 16 $\mathsf{\mu }$ L Laemmli buffer (ROTI^®Load 1, Carl Roth, Karlruhe, Germany) and incubated for 10 $\min$ at 96 °C. Then, 20 $\mathsf{\mu }$ L of sample and 10 $\mathsf{\mu }$ L molecular weight size marker (PageRuler™ 10 kDa to 180 kDa, Thermo Scientific™, Schwerte, Germany) were loaded on a precast polyacrylamid gel (12% Mini-PROTEAN^® TGX Stain-Free™ Protein Gels, Bio-Rad, Germany). As running buffer, a 1 to 10 dilution of ROTIPHORESE^®10x (Carl Roth, Karlsruhe, Germany) and ultrapure water was used. The gel separation was started using a voltage of 60 $\mathrm{V}$ for 30 $\min$ . The separation was continued at 150 $\mathrm{V}$ for a further 60 $\min$ . Afterwards, the gel was washed in water for 30 $\min$ and stained using ROTI^®Blue quick (Carl Roth, Karlsruhe, Germany) until protein bands became visible. For the gel image, the ChemiDoc XRS+ (Bio-Rad, Germany) was used.