Journal: STAR Protocols

Article Title: Optimized protocol for the identification of lipid droplet proteomes using proximity labeling proteomics in cultured human cells

doi: 10.1016/j.xpro.2021.100579

Figure Lengend Snippet: Schematic illustrating the major protocol steps for proximity biotinylation and proteomic analysis of lipid droplet proteins Cell lines expressing the APEX2 fusion proteins (i.e., Cyto-APEX2, PLIN2-APEX2, ATGL∗-APEX2) are treated to biotinylate endogenous LD proteins. LDs are then isolated by fractionation, biotinylated proteins are affinity purified, proteins are trypsinized, and peptides are analyzed by mass spectrometry.

Article Snippet: Rabbit polyclonal anti-PLIN2 , Abgent , Cat. # AP5118C; RRID: AB_10662954.

Techniques: Expressing, Isolation, Fractionation, Affinity Purification, Mass Spectrometry

Journal: STAR Protocols

Article Title: Optimized protocol for the identification of lipid droplet proteomes using proximity labeling proteomics in cultured human cells

doi: 10.1016/j.xpro.2021.100579

Figure Lengend Snippet: Characterization of APEX2 fusion protein expression, activity, and localization (A and B) Huh7 cells stably expressing Cyto-APEX2 and PLIN2-APEX2 were treated with the indicated amounts of doxycycline (dox) for 48 h and treated with biotin phenol and H 2 O 2 to induce biotinylation of endogenous proteins. The levels of protein expression and protein biotinylation were analyzed by western blotting with anti-V5 antibodies, anti-GAPDH antibodies, and streptavidin conjugated to IRDye 800CW. (C) Huh7 cells stably expressing Cyto-APEX2 and PLIN2-APEX2 were treated for 48 h with 5 ng/mL and 1 ng/mL doxycycline, respectively. Cells were then treated for 24 h with 200 μM oleate and 1 μM BODIPY-C12-568 (red). Cells were fixed in 4% paraformaldehyde, immunostained with anti-V5 antibodies, and imaged by fluorescence microscopy using a Deltavision Elite widefield epifluoresence deconvolution microscope (GE Healthcare). Scale bars represent 10 μm.

Article Snippet: Rabbit polyclonal anti-PLIN2 , Abgent , Cat. # AP5118C; RRID: AB_10662954.

Techniques: Expressing, Activity Assay, Stable Transfection, Western Blot, Fluorescence, Microscopy

Journal: STAR Protocols

Article Title: Optimized protocol for the identification of lipid droplet proteomes using proximity labeling proteomics in cultured human cells

doi: 10.1016/j.xpro.2021.100579

Figure Lengend Snippet: Proximity biotinylation and label-free quantification to define high confidence LD proteomes in Huh7 cells (A) Comparison of the iBAQ intensity and the previously determined spectral abundance factor ( Bersuker et al., 2018 ) for proteins identified using ATGL∗-APEX2 proximity labeling proteomics. The line of best fit is shown and the dotted lines indicate the confidence interval. (B) Comparison of the iBAQ intensity and the previously determined spectral abundance factor ( Bersuker et al., 2018 ) for proteins identified using PLIN2-APEX2 proximity labeling proteomics. The line of best fit is shown and the dotted lines indicate the confidence interval. (C) Comparison of the iBAQ intensity for proteins identified using PLIN2-APEX2 and ATGL∗-APEX2 proximity labeling proteomics. The line of best fit is shown and the dotted lines indicate the confidence interval. (D) Illustration of the Huh7 high confidence LD proteome determined using the methods described in this protocol. An asterisk indicates an LD protein that has been validated by imaging and was identified in our studies but was below the threshold.

Article Snippet: Rabbit polyclonal anti-PLIN2 , Abgent , Cat. # AP5118C; RRID: AB_10662954.

Techniques: Labeling, Imaging

Journal: STAR Protocols

Article Title: Optimized protocol for the identification of lipid droplet proteomes using proximity labeling proteomics in cultured human cells

doi: 10.1016/j.xpro.2021.100579

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-PLIN2 , Abgent , Cat. # AP5118C; RRID: AB_10662954.

Techniques: Recombinant, Modification, Protease Inhibitor, Avidin-Biotin Assay, Staining, Plasmid Preparation, Software