Bmper antibody

Isolated hepatocytes or LSECs were cultured on a coverslip (AGC Techno Glass Co., Shizuoka, Japan), fixed with paraformaldehyde for 5 min and blocked with SuperBlock blocking buffer (Thermo Fisher Scientific). The cells were incubated with an E-cadherin antibody (BD Biosciences, San Jose, CA, USA) and VE-cadherin antibody (Abcam) at 1:200 for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were used as secondary antibodies for 1 h at room temperature with protection from the light. The cells were counterstained with DAPI and analyzed by fluorescence microscopy (Keyence Corporation, Osaka, Japan).
Mouse livers were mounted in Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co Ltd., Tokyo, Japan) and frozen until preparation. Frozen sections were cut at 10-μm thickness and fixed in methanol for 10 min. Blocking was performed in PBS with 3 % bovine serum albumin (BSA). The slides were incubated with BMPER antibody (Abcam) at 1:200 in PBS overnight at 4 °C, and subsequently incubated with CD31 antibody (BD Pharmingen) at 1:100 in PBS for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were incubated at 1:200 in PBS for 1 h at room temperature. The slides were counterstained with DAPI and analyzed using fluorescence microscopy.

Immunoprecipitation of mouse plasma was performed using Dynabeads (Life Technologies), as follows. Dynabeads were incubated with 2 μg of BMPER antibody (Abcam) or rabbit IgG (Abcam) for 1 h at room temperature. A total of 10 μL of plasma was precipitated for each sample. The samples were mixed with SDS buffer, and BMPER and BMP6 expression was analyzed by western blot.