Imager 600 system

Frozen RV was homogenized using ice-cold lysis buffer and proteinase inhibitor cocktail (Manchini et al., 2014 (link)). Lysates corresponding to 30 μg of protein were subjected to 10% SDS-PAGE. Separated proteins were transferred to PVDF membrane (Amersham Biosciences, NJ, USA) and transfer effectiveness was examined with 0.5% Ponceau S. After blocking with 5% non-fat dry milk for 2 h at room temperature, PVDF membranes were probed with Abcam (Cambridge, MA, USA) primary antibodies for rabbit ant-Akt₁ (1:5000), rabbit anti-p-Akt₁ (1:2500), rabbit anti-Caspase3; rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:1000), rabbit anti-Bcl-xL (1:500), rabbit anti-β-MHC (1:5000), rabbit anti-α-MHC (1:5000), rabbit anti-L-type Ca⁺⁺ (1:500), rabbit anti-ryanodine receptor (1:1000), rabbit anti-Serca 2 (1:1000), and rabbit anti-Na⁺/Ca⁺⁺ exchanger (1:100) in overnight incubation. Membranes were then washed five times with PBS and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit (1:20,000; Zymed, CA, USA). Membranes were again washed five times with blocking buffer and then rinsed twice with PBS. Antibodies binding were detected by chemiluminescence reagents (Amersham Biosciences, NJ, USA), and images were captured using an Amersham Imager 600 system. Quantification of target proteins was normalized for the internal control glyceraldehyde 3-phosphate dehydrogenase.

The protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring onto nitrocellulose filter membrane (Biosharp, 20030). The membranes were blocked with 5% skim milk in tris-buffered saline with 0.1% tween 20 (TBST) for 2 h and then incubated with the anti-N (1:1000), anti-P (1:1000), anti-M (1:1000), anti-G (1:1000), anti-L (1:1000), anti-hnRNPA1 (1:1000), anti-Flag (1:2000), anti-Myc (1:4000), anti-HA (1:4000), anti-EGFP (1:2000), anti-Histone H3 (1:4000), or anti-β-actin (1:5000) antibodies for 4 h. After three-time washing with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5000) or anti-mouse antibody (1:5000) for 1 h. The signal intensity was visualized using Amersham Imager 600 System after washing with TBST for three times.

To analyze ERα protein expression, total protein from each sample was first isolated from L3-L5 vertebrae by homogenization with RIPA buffer (Solarbio) followed by sonication. Protein concentrations were found for lysates using BCA assay. For ERα protein expression in Raw 264.7 cells (+/− 20 ng/ml RANKL) cells were cultured onto 6 well tissue culture plates with either control, 1 μM ZA or 100 ng/ml rmCst6 treatments and incubated at 37° C for 48 hr. (2.5 × 10⁵ cells/ml, 2 ml total volume). After 48 hr. cells were lysed using RIPA buffer and protein concentration was found using BCA assay. Both protein tissue and protein cell samples were prepared for SDS PAGE, equal amount of protein were loaded for each sample and then ran on 10% Acrylamide/Bis-acrylamide gels (N = 6 for bone tissue, N = 3 for Raw 264.7 cells). Following membrane transfer, mouse anti h/m/r ERα (MAB57151, R&D Systems) was used as the primary antibody and HRP conjugated anti-mouse IgG (R&D systems, HAF018) was used as the secondary antibody. Bands of interest were visualized and imaged under chemiluminescent detection using the Amersham Imager 600 System. Amido black staining (for tissue lysate samples) and mouse anti β-actin antibody (Sigma, A1978) (for cell lysate samples) were used as loading controls for western blots. Densitometry analysis of western blot bands was performed using ImageJ.