Journal: eLife

Article Title: Aging impairs cold-induced beige adipogenesis and adipocyte metabolic reprogramming

doi: 10.7554/eLife.87756

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti–red fluorescent protein (RFP) , Rockland, Pottstown, PA , 600-401-379, RRID: AB_2209751 , 1:500.

Techniques: Sequencing, Isolation, Sensitive Assay, Software, Extraction, Staining, Cell Culture, SYBR Green Assay, Lysis

Journal: eLife

Article Title: Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis

doi: 10.7554/eLife.82821

Figure Lengend Snippet: ( A ) Islet extracts (40 μg) from WT, MlphKO, Exo8KO, and ME8DKO mice were immunoblotted with antibodies toward the indicated proteins (left). Protein expression levels were quantified by densitometric analyses from 3 to 5 independent preparations (right). ( B ) MIN6 cells were infected with adenoviruses encoding control LacZ, mCherry-exophilin-8, and/or FLAG-melanophilin. After 2 days, the cell extracts underwent immunoprecipitation with mCherry nanobody. The immunoprecipitates (IP), as well as the 1% extracts (Input), were immunoblotted with anti-FLAG and anti-red fluorescent protein (RFP) antibodies. ( C ) MIN6 cell extracts were immunoprecipitated with rabbit anti-melanophilin antibody or control IgG, and the immunoprecipitates were immunoblotted with antibodies toward the indicated proteins. ( D ) HEK293A cells cultured in 10 cm dishes were transfected with mCherry-fused, exophilin-8 (left) or melanophilin (right) with the indicated EGFP-fused exocyst components. After 48 hr, the cell lysates were subjected to immunoprecipitation with mCherry-nanobody-bound glutathione-Sepharose beads. EGFP and mCherry fluorescence on the precipitated beads was observed by confocal microscopy. ( E ) MIN6 cells expressing FLAG-tagged, melanophilin or exophlin-8 were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were immunoblotted with antibodies toward the indicated proteins. Note that the exocyst complex components exist in both immunoprecipitates, whereas RIM-BP2, RIM2, myosin-VIIa, and myosin-Va largely exist only in one of them. Bars, 100 μm. ###p<0.001 by one-way ANOVA. Figure 3—source data 1. Uncropped blot images of .

Article Snippet: Antibody , Anti-red fluorescent protein (RFP) (rabbit polyclonal) , MBL , Cat#: PM005; RRID: AB_591279 , WB (1:10,000).

Techniques: Expressing, Infection, Control, Immunoprecipitation, Cell Culture, Transfection, Fluorescence, Confocal Microscopy

Journal: eLife

Article Title: Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis

doi: 10.7554/eLife.82821

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-red fluorescent protein (RFP) (rabbit polyclonal) , MBL , Cat#: PM005; RRID: AB_591279 , WB (1:10,000).

Techniques: Transfection, Construct, Isolation, Recombinant, Plasmid Preparation, Software, Western Blot, Mass Spectrometry

Journal: eLife

Article Title: Protein composition of axonal dopamine release sites in the striatum

doi: 10.7554/eLife.83018

Figure Lengend Snippet: ( A ) Western blots of striatal lysates (input) and the pellet of the affinity-purified fractions (pulldown) are shown. The striata were harvested from mice that were injected with AAVs expressing BirA-tdTomato in dopamine neurons, or from uninjected control mice, with or without subcutaneous injection of biotin. For a timeline of the experiment, see . BirA-tdTomato was detected with anti-red fluorescent protein (RFP) antibodies. The RFP bands, reflecting self-biotinylated BirA-tdTomato, were only detected in mice subcutaneously injected with biotin, but not when biotin was not supplied or when BirA-tdTomato was not expressed. β-Actin was present in the brain input, but was depleted during affinity purification. BirA-tdTomato expression is too low to be detected in the input given that dopamine axons account at most for a few % of the striatal volume. The full-length BirA-tdTomato protein has a molecular weight of 91 kD. Because the smaller molecular weight bands detected with the anti-RFP antibodies (marked with *) are only present when BirA-tdTomato is enriched, they are likely degradation products. Western blots are from a single qualitative pilot experiment, 2 striata per condition. ( B ) Same as ( A ), but in mice expressing ELKS2β-BirA. ELKS2β-BirA is detected with anti-HA antibodies; Western blots from a single qualitative pilot experiment, 4 striata per condition. These experiments reveal that BirA fusion proteins are self-biotinylated in vivo after biotin injections and are purified with iBioID. The purification is specific enough to reduce contamination by strongly expressed proteins present in all cells, for example, β-actin. For unedited scans of Western blots, see . Figure 1—figure supplement 2—source data 1. Western blots for . ( A ) Original scan (5 min) of anti-RFP Western blots shown in . ( B ) Grayscale scans (left) and brightness and contrast-adjusted scans (right) of Western blots shown in . Stars denote bands that are likely degradation products of the BirA-tdTomato fusion protein. Arrows denote bands that are likely cross-reactive; they are present independent of BirA-tdTomato expression and are strong in intensity because a long exposure is shown and a large amount of input was loaded. ( C ) Original scan (3 s) of anti-β-actin Western blots shown in . ( D ) Grayscale scans (left) and brightness and contrast-adjusted scans (right) of Western blots shown in . Arrowheads denote bands that likely represent synapsin isoforms. Figure 1—figure supplement 2—source data 2. Western blots for . ( A ) Original scan (5 min) of anti-HA Western blots shown in . ( B ) Grayscale scans (left) and brightness and contrast-adjusted scans (right) of Western blots shown in .

Article Snippet: Antibody , Anti-red fluorescent protein (rabbit polyclonal) , Rockland , CAT#600-401-379; RRID: AB_2209751 , LAC A81 , WB (1:1000).

Techniques: Western Blot, Affinity Purification, Injection, Expressing, Control, Molecular Weight, In Vivo, Purification

Journal: eLife

Article Title: Protein composition of axonal dopamine release sites in the striatum

doi: 10.7554/eLife.83018

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-red fluorescent protein (rabbit polyclonal) , Rockland , CAT#600-401-379; RRID: AB_2209751 , LAC A81 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Immunofluorescence, Western Blot, Software