Phospho rb

Total protein extract was obtained by adding to the cells RIPA buffer supplemented with protease inhibitor (PMSF, Sigma-Aldrich, and cOmplete, Roche, Basel, Switzerland) and phosphatase inhibitor (PhosSTOP, Roche Basel, Switzerland). Protein concentrations were measured by BCA protein assay kit (Pierce Biochemicals Waltham, MA, USA) following manufacturer instructions and 50 µg of protein extract was electrophoresed on an 8% polyacrylamide gel and transferred on a nitrocellulose membrane (GE Healthcare Chicago, IL, USA). Primary antibodies: RB (Abcam, ab181616), Phospho-RB (SantaCruz, sc-271930), E2F3A (SantaCruz, sc-56665), MYCN (SantaCruz, sc-53993), HA (Cell Signaling, #3724), GAPDH (10494-1-AP ProteinTech, Rosemont, IL, USA), β-tubulin (ProteinTech 66240-1-IG), β-actin (Sigma-Aldrich, A2228). Secondary antibodies: rabbit anti-mouse and goat anti-rabbit HRP (Jackson, 115-035-003, 111-035-144). Membranes were incubated with Clarity Western ECL (BIO-RAD, Hercules, CA, USA) and then scanned with ChemiDoc (BIO-RAD).

Fifty μg of protein was subjected to western blot analysis [62 (link)]. Blots were incubated overnight at 4°C with antibodies against GPER, Cyclin E (CCNE), Cyclin B1 (CCNB1), phospho-Rb, Cytochrome c, Bax, Bcl-2, Parp1, pERK1/2-ERK2 (all from Santa Cruz Biotechnology, Santa Cruz CA, USA). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) and immunoreactive bands were visualized with the ECL western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ). To assure equal loading of proteins, membranes were stripped and incubated overnight with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology).