perilipin3  (Novus Biologicals)

Journal: Cell reports

Article Title: RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal

doi: 10.1016/j.celrep.2021.109451

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-TIP47 (F-10) (Perilipin3) , Santa Cruz Biotechnology , Cat# sc-390968.

Techniques: Recombinant, Software, Microscopy

Journal: eLife

Article Title: Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting

doi: 10.7554/eLife.01607

Figure Lengend Snippet: ( A ) The endogenous COPI machinery stained with αCOP or garz antibodies (red) localizes to LDs in S2 cells. Frequencies of colocalization of αCOP and garz spots with LDs from experiments are higher than expected from a random distribution. ( B ) The endogenous COPI machinery localizes to LDs in NRK cells. NRK cells stained for βCOP or GBF1 by immunofluorescence (red) show partial colocalization with LDs stained with BODIPY (green). Colocalization of βCOP with LDs in NRK cells is not random. Relative frequencies of βCOP, KDEL receptor and clathrin spots colocalizing with LDs determined in experiments are respectively compared to the frequencies of colocalization from a binomial random distribution. From the two frequencies (experiment vs simulation), a significant overrepresentation of βCOP on LDs is observed, whereas clathrin and KDEL receptor (KDELR) are not found on LDs. For ( A ) and ( B ) scale bars are 10 μm (overview) or 1 μm (first inlay) or 250 nm (second inlay). Statistical significance was tested by a student t test with p<0.01 (n = 30) . ( C ) Localization of β’COP (green) to the LD surface (perilipin3, red) using confocal (upper panel) and super-resolution STED microscopy (lower panel). Scale bar = 500 nm (overview) or 100 nm (inlay). ( D ) Localization of β’COP to LDs is efficiently blocked by treatment of cells with the Arf1 GEF inhibitors brefeldin A or golgicide A . Scale bar = 10 μm (overview) or 1 μm (inlay). DOI: http://dx.doi.org/10.7554/eLife.01607.008

Article Snippet: Rabbit polyclonal antibodies used: anti-GPAT4 , anti-CCT1 , anti-GBF1 (BD Biosciences, San Jose, CA), anti-KDEL-receptor (KDELR; gift from Dr JE Rothman; Yale University), anti-βCOP (gift from Dr JE Rothman; Yale University), anti-perilipin3 (TIP47; Novus Biologicals, Littleton, CO), anti-αCOP (Abcam, Cambridge, MA), anti-GRP78/BiP (ET-21) (Sigma–Aldrich, St. Louis, MO) and anti-garz ( ) (gift from Dr A Paululat; University of Osnabrück).

Techniques: Staining, Immunofluorescence, Microscopy

Journal: eLife

Article Title: Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting

doi: 10.7554/eLife.01607

Figure Lengend Snippet: ( A ) The probability P of having n colocalization events from nA type A dots (A = βCOP, αCOP, clathrin, KDELR) follows a binomial distribution. ns is the fraction with the highest probability of colocalization from the random distribution. nexp is the fraction of colocalization observed in the experiment. If nexp is larger than ns, A is overrepresented on LDs. A representative plot of P(n) obtained for an experiment of βCOP colocalization with LDs is depicted showing that β COP is overrepresented on LDs (nexp >> ns). ( B ) Arf79F-cherry (red) colocalizes with the LD marker protein CGI-58 (green). LDs are visualized by MDH (blue). ( C ) GFP-GM130 (green) and Arf79F-cherry (red) show colocalization in the perinuclear region of S2 cells, representing the Golgi apparatus (lower panel) but not on LDs (upper panel). ( D ) Arf79F-GFP binding to LDs is stabilized by treatment with brefeldin A . Arf79F-GFP localization was followed after the addition of 100 µM brefeldin A over time. LDs are stained with LipidTOX. Left panels show representative images of such an experiment. Right panel shows a quantification of at least three independent experiments. Error bars represent the s.d. of the mean. Scale bars are 10 μm (overview) or 1 μm (inlay). ( E ) NRK cells stained for β’COP by immunofluorescence (red) show partial colocalization with LDs stained with BODIPY (green). ( F ) NRK cells costained for endogenous βCOP (red) and β’COP (green) show colocalization in the same structures on LDs visualized by MDH (blue). ( G ) NRK cells co-stained for endogenous clathrin (green) and βCOP (red) (upper panel) or β′COP (green) and KDELR (red) (lower panel) show no colocalization on LDs (blue). ( H ) Endogenous β’COP (green) colocalizes with the LD marker protein perilipin3 (red). LDs are visualized by MDH (blue). ( I ) NRK cells co-stained for endogenous GM130 (green) and βCOP (red) show colocalization in the perinuclear region of cells, representing the Golgi apparatus (lower panel) but not on LDs (upper panel). DOI: http://dx.doi.org/10.7554/eLife.01607.009

Article Snippet: Rabbit polyclonal antibodies used: anti-GPAT4 , anti-CCT1 , anti-GBF1 (BD Biosciences, San Jose, CA), anti-KDEL-receptor (KDELR; gift from Dr JE Rothman; Yale University), anti-βCOP (gift from Dr JE Rothman; Yale University), anti-perilipin3 (TIP47; Novus Biologicals, Littleton, CO), anti-αCOP (Abcam, Cambridge, MA), anti-GRP78/BiP (ET-21) (Sigma–Aldrich, St. Louis, MO) and anti-garz ( ) (gift from Dr A Paululat; University of Osnabrück).

Techniques: Marker, Binding Assay, Staining, Immunofluorescence