gfp sec61β 121159  (Addgene inc)

Journal: STAR Protocols

Article Title: In vitro and in vivo assay of the ER lipid scramblase TMEM41B

doi: 10.1016/j.xpro.2022.101333

Figure Lengend Snippet: In vivo scrambling activity assay showing TMEM41B shuttles PC between the ER membrane leaflets (A) Schematic of metabolic labeling and click-chemistry to detect PC in Huh7 TMEM41B WT/KO cells. Cells are incubated with alkyne-choline for synthesis into alkyne-PC at the ER outer leaflet. In wild-type cells, alkyne-PC will shuttle across the bilayer into the inner leaflet of the ER. Digitonin can permeabilizing the cell surface membrane, but the endo-membrane would be intact. Therefore, the click-chemistry reagents could only label alkyne-PC on the outer leaflet of the ER but could not access the inner leaflet of ER membrane. The absence of scramblases in the ER of mutant cells would trap more alkyne-PC on the outer leaflet and increase the fluorescent signal. (B) Representative western blots for validation of TMEM41B deficiency in Huh7 cells. (C) Representative confocal images showing accumulation of alkyne-PC signal on the outer leaflet of the ER in the absence of TMEM41B (n=3 independent experiments). CRISPR/Cas9-mediated control or TMEM41B KO Huh7 cells incubating with alkyne-choline are clicked with 5-TAMRA azide and visualized by confocal microscopy. The ER is marked by GFP-SEC61β. The middle image is a zoomed in image from the white box in the left image. Right: surface plots depicting alkyne-PC signals. Scale bars, 5 μm. (D) Quantification of colocalization of signals of alkyne-PC and GFP-SEC61β (left) and total fluorescence signals (a.u.) of alkyne-PC (right) from (B). Data are presented as mean ± SEM. n.s., no significance, ∗∗∗∗p < 0.0001 (two-tailed Student’s t test).

Article Snippet: Plasmid: GFP- SEC61β , , Addgene Cat# 121159.

Techniques: In Vivo, Activity Assay, Membrane, Labeling, Incubation, Mutagenesis, Western Blot, Biomarker Discovery, CRISPR, Control, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: STAR Protocols

Article Title: In vitro and in vivo assay of the ER lipid scramblase TMEM41B

doi: 10.1016/j.xpro.2022.101333

Figure Lengend Snippet:

Article Snippet: Plasmid: GFP- SEC61β , , Addgene Cat# 121159.

Techniques: Virus, Recombinant, Protease Inhibitor, Gel Extraction, Plasmid Preparation, Software, Cell Culture, Molecular Weight, Membrane, Blocking Assay, Spectrophotometry, Evaporation