Anti bmal1 antibody

OCN and Bmal1 double immunofluorescence staining was performed to evaluate the expression of BMAL1 in osteoblasts. Briefly, decalcified femora were dehydrated in 30% sucrose, embedded in OCT compound and cut into 5-μm-thick sections. After blocking with 5% donkey serum at room temperature for 30 min, the samples were incubated with an anti-BMAL1 antibody (1:100; Abcam; Britain) and an anti-OCN antibody (1:100; Abcam; Britain) overnight at 4 °C. Double-immunofluorescence staining was performed with a Treble-Fluorescence Immunohistochemical Mouse/Rabbit kit (Immunoway, USA) according to the manufacturer’s protocol. The number of BMAL1⁺ osteoblasts on the trabecular bone surface was calculated in three sections per mouse using ImageJ v1.52 software. The proportion of BMAL⁺ osteoblasts to total osteoblasts was also calculated.

Nuclear extracts were made from liver according to previously published protocol (52 (link)). Protein concentrations were determined by Bradford assays (Bio-Rad), and aliquots were snap frozen in liquid nitrogen and stored at −80°C until usage. Immunoblot analyses were performed as described previously (53 (link)). Briefly, 25 μg of proteins separated by 4 to ~20% gradient SDS–polyacrylamide gel electrophoresis gels (Bio-Rad) were transferred to nitrocellulose membranes, blocked in TBST buffer supplemented with 5% bovine serum albumin or 5% fat-free milk, and incubated overnight with primary anti-SON antibody (Abcam, 121033), anti-BMAL1 antibody (Abcam, 3350), anti-PERK (Cell Signaling Technology, no. 3192), anti–phospho-PERK (Thr⁹⁰⁸) (Thermo Fisher Scientific, MA5-15033), anti-ATF4 (Cell Signaling Technology, no. 11815), anti-IRE1α (Cell Signaling Technology, no. 3294), anti–phospho-IRE1α (Ser⁷²⁴) (ABclonal, AP0878), anti-XBP1s (BioLegend, 658802), anti-ATF6 (Santa Cruz Biotechnology, sc-166659), and β-actin (Cell Signaling Technology, no. 12620) at 4°C. Blots were incubated with an appropriate secondary antibody coupled to horseradish peroxidase at room temperature for 1 hour and reacted with Enhanced chemiluminescence (ECL) reagents per the manufacturer’s (Thermo Fisher Scientific) suggestion and detected by the Bio-Rad ChemiDoc MP Imaging System.