Tissue protein extract reagent

Skin tissue was snap frozen in liquid nitrogen, and protein lysates were obtained using Tissue Protein Extract Reagent and Halt Protease and Phosphatase Inhibition Cocktail (Thermo Fisher Scientific). After homogenization, lysates were spun down for 10 min at 4°C and 14,000g. The supernatants were collected, and protein content was quantified using a bicinchoninic acid assay (BCA assay, Thermo Fisher Scientific). Normalized samples were then analyzed for CCL21 content by ELISA (cat. DY457, R&D Systems).

ICR mice were housed under controlled temperature conditions (22 ± 2ºC) in an artificially illuminated room (12h-light/12h-dark). Food and tap water were
available ad libitum. Females were mated with males at 70–90 days. The day of birth was designated as day 0. At 8–9 weeks, the female mice were
ovariectomized (OVX) under isoflurane anesthesia. A silastic tube (Kaneka, Tokyo, Japan) containing 10 mg of 17β-estradiol (E2; TOKYO CHEMICAL INDUSTRY, Tokyo,
Japan) was subcutaneously implanted into OVX female mice 7 days after OVX and the tube was left in place for 7 days. Control mice were treated with an implanted
silastic tube without E2. After 7 days anterior pituitary glands were collected and lysed in Tissue-Protein Extract Reagent (Thermo Fisher Scientific, Waltham,
MA, USA) to extract the protein. The total RNA was extracted from the anterior pituitary glands using an RNeasy mini Kit (Qiagen, Venlo, The Netherlands) and
reverse-transcribed into cDNA using a QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s protocol.