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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2013 Dec 19;34(2):386–396. doi: 10.1161/ATVBAHA.113.302448

Figure 3. LDAH is a serine hydrolase that plays a role in cholesterol homeostasis.

Figure 3

(A) HeLa cells were transfected with flag, flag-mLDAH, or flag-(S140->C)-mLDAH. Cell lysates were incubated with the ActivX® DTB-FP to label active serines in serine hydrolases. Proteins bound to the probe were pulled-down using streptavidin-agarose. The streptavidin-captured fraction and total cell lysates were immunoblotted with anti-flag. (B) In vitro CE hydrolase activity performed on protein extracts from HeLa cells transfected with flag, flag-mLDAH, or flag-(S140->C)-mLDAH. (n=3). (C) HEK293 cells were transfected with flag, flag-mLDAH, or flag-(S140->C)-mLDAH. The cells were cultured in DMEM-10% FBS and remained untreated (−CHOL), or were treated with CHOL:MβCD (10 μg/ml) for 24h (+CHOL). Total intracellular cholesterol, FC and CE levels were quantified and normalized to protein. (n=3). Immunoblots with anti-flag on protein lysates from the cells used for the experiments are shown under the chart. (D) Cholesterol levels in untreated or cholesterol-loaded HEK293 cells transfected with non-target or hLDAH siRNAs. (n=3). Immunoblots with anti-hLDAH on protein lysates from the cells used for the experiments are shown under the chart. (E) Immunofluorescence with an anti-flag antibody (green) on HEK293 cells transfected with flag-mLDAH and flag-(S140->C)-mLDAH; LipidTox™ (red) was used to stain LDs. All data are shown as mean ± SD. *p<0.05, **p<0.01 vs. flag-mLDAH or non-target-siRNA.