Mc 20

Muscle protein lysates (60 µg/lane) were resolved by SDS-PAGE, and proteins were transferred onto nitrocellulose membranes, according to standard procedures. Equal loading and transfer efficiency were verified by Ponceau S Red (Sigma) staining. Membranes were blocked for 1 h in 1X TBST (20 mmol/L Tris-base, 150 mmol/L NaCl, 0.1% Tween-20, pH 7.5) containing 5% skim milk powder and 3% bovine serum albumin (BSA), and incubated overnight with primary antibody. Primary antibodies used were: ERα (1:500, MC-20, Santa Cruz; 1:1000 [E115] Abcam ab32063), ERβ (1:750, ab3576, Abcam), PIK3C2B (1:1000, Clone 22/PI3-K, BD Biosciences), DNM2 (1:500 Clone 2862, Dr. Jocelyn Laporte, IGBMC France), DNM2 (1:1000 G-4, Santa Cruz), β-actin (1:5000, #4967 Cell Signaling; 1:5000 Abcam ab8226) and HSP90 (Clone OTI4C10, Origene). After extensive washing in 1X TBST, membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:5000, BioRad) in 1X TBST containing 5% skim milk powder and 3% BSA.

Tissue biopsies were fixed in 3.7% formaldehyde, embedded in paraffin and processed for histological examination. Histology was determined by a pathologist (LK) from 4 µm haematoxylin & eosin (Sigma-Aldrich, Zwijndrecht, The Netherlands) stained sections. Detection of estrogen receptor-α (ERα) of human origin was performed with immunohistochemistry using the antibody HC-20 (Santa Cruz Biotechnologies, Heidelberg, Germany), whereas for mouse ERα, antibody MC-20 (Santa Cruz Biotechnologies) was used. In brief, sections were subjected to deparaffinization followed by rehydration. Heat-induced antigen retrieval was performed and slides were blocked in 1% BSA/PBST and subsequently incubated overnight at 4 °C with 500 times diluted ERα antibody, as described earlier [10 (link)]. The EnVision detection system was used according to the manufacturer’s manual followed by visualization with Diaminobenzidine (DAB).

Western Blot and protein preparation of uteri and bone from NOER and WT mice were performed as previously described24 (link). Briefly, tissues were homogenized in RIPA-buffer supplemented with complete Mini EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics). The rabbit polyclonal ERα antibody (MC-20; Santa Cruz Biotechnology), diluted 1:1000, was used24 (link). An anti-rabbit HRP-conjugated secondary antibody (GE Healthcare), diluted 1:10,000, and Clarity Western ECL substrate (BioRad), were used to visualize the bands.