Journal: Cell reports

Article Title: Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System.

doi: 10.1016/j.celrep.2017.05.028

Figure Lengend Snippet: Figure 1. CRISPR Knockout of ORP1L in HeLa Cells Reduces Cholesterol Esterification in the ER (A) Domain organization of ORP1L and ORP1S and location of the guide RNA (gRNA)-targeting site in OSBPL1 (Ank, ankyrin motif). (B) RT-PCR analysis of ORP1L mRNA expression. (C) Immunoblot analysis of ORP1L and ORP1S expression in HeLa and ORP1L-null cells. (D and E) [3H]Oleate incorporation into cholesterol ester (D) and TAGs (E) in HeLa, ORP1L-null, and ORP1L-null cells transiently expressing an ORP1L cDNA (ORP1L rescue) and cultured in FBS or LPDS for 16 hr (*p < 0.0001). (F) Lysates of cells from (D) were immunoblotted for ORP1. (G) Cholesterol esterification in cells cultured in FBS and treated with no addition (NA, solvent) or 25-hydroxycholesterol (25OH, 6 mM) for 2 hr (*p < 0.05 and **p < 0.001). (H) Lysates from cells cultured in FBS (+) or LPDS (), as described in (A), were probed with an antibody against human ACAT1. (I) HeLa and ORP1L-null cells were cultured as described in (A) and received no addition (NA, solvent) or brefeldin A (BFA, 5 mg/mL) for 1 hr prior to measuring cholesterol esterification (*p < 0.01 and **p < 0.0001). Results of experiments in (D), (E), (G), and (I) are the mean and SD of three to four experiments.

Article Snippet: Protein expression was analyzed using primary antibodies against human ACAT1 (1:1,000, rabbit polyclonal raised against amino acids 9–113), pan-OSBP/ORP (1:15,000, rabbit polyclonal raised against the C terminus of ORP2), Rab7 (1:2,000, Cell Signaling Technology, D95F2), NPC1 (1:2,000, Abcam, ab36983), ORP1L (1:2,000, Abcam, ab131165), LAMP1 and LAMP2 (1:1,000, Developmental Studies Hybridoma Bank, 1D4B and H4B4), LDLR (1:2,000, rabbit polyclonal Ab3143 targeting the C terminus) (Russell et al., 1984), or actin (1:10,000, Sigma, AC15).

Techniques: CRISPR, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Solvent