Digital imaging system

Levels of mitochondrial ERα from control, MPP-, estradiol-, and MPP + estradiol-treated rat calvarial osteoblasts were immunodetected as described previously [40 (link)]. When preparing mitochondrial proteins, a mixture of proteinase inhibitors, including 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 5 µg/mL leupeptin, was added to the buffer to prevent protein degradation by proteinases. Protein concentrations were quantified using a bicinchonic acid protein assay kit (Thermo, San Jose, CA, USA). Mitochondrial proteins (100 μg/well) were subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). After that, proteins separated on the polyacrylamide gel were electronically transferred onto nitrocellulose membranes. After being blocked with 5% non-fat milk at 37 °C for 1 h, levels of ERα on the membrane were immunodetected using a rabbit polyclonal antibody (pAb) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HSP-60 was immunodetected as an internal control of mitochondrial proteins. Intensities of the immunoreactive protein bands were determined using a digital imaging system (Syngene, Cambridge, UK) and densitometry software (Syngene) as described previously [41 (link)].

Cell cultures were lysed with RIPA lysis buffer (Tris-HCl 10 mmol/L, EDTA 5 mmol/L, NaCl 50 mmol/L, 1% deoxycholic acid and 1% triton X-100, pH 7.4). Protein concentration was determined by the Bradford method (BioRad protein assay). Equal amounts of proteins from cell extracts were separated by SDS-PAGE 8–15%, electro-transferred to PVDF membranes and blocked with 5% milk. Primary antibodies were used against type I collagen 1:2000 (cat #ABT123 Millipore), p62 1:2000 (cat #5114 Cell Signaling), osteopontin 1:2000 (cat #ab8448 Abcam), BECLin1 1:2000 (cat #3738 Cell Signaling), α-SMA 1:20000 (cat #ab7817 Abcam), GAPDH 1:50000 (cat #8795 Sigma), SM22 1:10000 (cat #ab14106 Abcam), LC3-I/II 1:1000 (cat #2775 Cell Signaling). Membranes were re-blotted with a horseradish peroxidase-linked secondary antibody 1:5000 (mouse cat #402335 and rabbit cat#401315 Merck). Bands were detected using ECL (Biological Industries) and luminescence was assessed using a digital imaging system (Syngene). Quantification of the bands by densitometry was performed using UN-SCAN-IT gel software.

Vascular smooth muscle cells were lysed using the RIPA lysis buffer (Tris-HCl 10 mmol/L, EDTA 5 mmol/L, NaCl 50 mmol/L, 1% deoxycholic acid and 1% Triton X-100, pH 7.4). Protein concentration was determined in A7r5 line by Bradford method (BioRad protein assay) while in RASMCs, protein concentration was determined by the bicinchoninic acid assay (Pierce BCA protein assay, Thermo Scientific). Equal amounts of protein from cells were separated by 7–15% SDS polyacrylamide gel electrophoresis and electrotransferred to PVDF membranes and blocked with 5% defatted milk in Tris-buffered saline pH 7.6, containing 0.1% (v/v) Tween 20 (TBS-T). Membranes were incubated with the primary antibodies at 4°C overnight. Membranes were then incubated with horseradish peroxidase-linked secondary antibody in 1% (w/v) defatted milk in TBST. The bands were detected using ECL (cat # NEL103001EA, Perkin Elmer, Waltham, MA, United States) luminescence was assessed using a digital imaging system (Syngene). Quantification of the bands by densitometry was performed using UN-SCAN-IT gel software. Protein content was normalized by β-tubulin or GAPDH.