Isolating and Quantifying Membrane-Bound Proteins

Human monocytes were lysed in RIPA buffer to extract the whole cell lysate or lysed following the two-step membrane-bound protein isolation protocol^{74 (link)} for CH25H detection. The lysates were subjected to western blot analysis. Briefly, blotted membrane was blocked with 5% BSA (Sigma) containing PBS for 1 h, incubated with primary antibodies (Abs) overnight, and incubated with secondary Abs in blocking buffer. β-actin (Sigma, clone AC-15) and Na, K-ATPase 1 protein (CST, #3010 S) was served as loading control, respectively. ATF3 and CH25H were detected using anti-ATF3 Ab (CST, clone: D2Y5W) and anti-CH25H Ab (Invitrogen, #PA5-72329), respectively, and IRDye 680RD donkey anti-rabbit secondary antibody (LI-COR Bioscience). Results were analyzed using a LI-COR CL Odyssey imager.
For analyses of cholesterol and 25HC levels, DCs were isolated from spleen of indicated mice and cell lysate was made using lysis buffer from Promega Cholesterol/Cholesterol Ester-Glo assay kit (#J3190) or by multiple thaw-freeze cycles. ELISA-based analyses of the levels of cholesterol or 25HC was carried out using Cholesterol/Cholesterol Ester-Glo assay ELISA kit (Promega, #J3190) or Mouse 25-hydroxycholesterol ELISA kit (MyBioSource, # MBS7256104) according to the manufacturers’ recommendations.