Fig 1: Labeling of secreted proteins and their detection in distal organs.a Schematic of the biotinylated Drosophila insulin-like peptide 2 (Dilp2) experiment. BirA*G3-ER (endoplasmic reticulum) was expressed in the insulin-producing cells (IPCs). b BirA*G3-ER+ (red), Biotin+ (white), Dilp2-HA+ (green)11 cell bodies, and BirA*G3-ERnegative, biotin+ and Dilp2-HA+ parts of the cell body (blue arrowhead) and ventral projections (which include dendrites and axons, which extend further13; white arrowhead) were detected. Scale bar: 2 µm. c Spatially informed bead enzyme-linked immunosorbent assay (ELISA) of biotinylated Dilp2-HA trafficking from head. Data are mean ± SEM absorbance (Abs.). Top: n = 2 biological replicates. Std. means standard peptide (1.04 × 10-11 M standard biotin-Flag-GS-HA peptide (n = 1)). The mean of Dilp215H>BirA*G3-ER-myc body is shown with the red line. Bottom: n = 3 biological replicates; two-tailed t-test (#p = 0.08996). N.S. means not significant. Approximate locations of IPC axonal projections in the cartoon diagram are based on ref. 14. d Fat body (FB)-expressed LPP-Gal4>BirA*G3-ER-myc is detected in abdomens but not legs, and muscle-expressed MHC-Gal4>BirA*G3-ER-myc is detected in thoraxes but not heads. Middle panel: higher exposure of the myc blot. IB: immunoblot. See Supplementary Fig. 2bb for the parts of legs used. For each slice, all lanes are from the same blot (see Source Data for uncropped blots). d' Quantification of signals from n = 3 independent experiments. Statistics: mean ± SEM integrated density of myc signals, normalized to tubulin (Tub) staining; one-way ANOVA and Benjamini, Krieger, Yekutieli linear two-stage step-up FDR. **1p = 0.0038; **2p = 0.0035; N.S. (p = 0.51). e Streptavidin bead pulldown followed by streptavidin-HRP detection in the leg and head lysates after FB and muscle biotinylation using BirA*G3-ER or BirA*G3 (cytoplasmic/nuclear for unconventionally secreted proteins). All lanes are from the same blot (see Source Data for uncropped blots). Representative result of three western blots. In d, e MHC>BirA*G3-ER and LPP>BirA*G3-ER are shown in color for clarity. In b–e flies were maintained with 50 µM biotin in food during adulthood. wt means wild-type. See also: Supplementary Figs. 1–3. Source data are provided as a Source Data file.
Fig 2: Rosa26 (R26) BirA*G3–ER and R26 BirA*G3–ER;Cre 3A mouse embryonic stem cell (mESCs) characterization.a Schematic showing Cre-induced recombination of the R26 BirA*G3-ER allele to generate the BirA*G3-ER;Cre expressing cell line. GFP is in blue, BirA*G3-ER-myc is in black, mKate2 is in red, homologous regions to the R26 locus are in blue, and other elements are colored in gray. b–i Native immunofluorescence in BirA*G3-ER parental (GFP+ (green)/mKate2-) or Cre-recombined (GFP-/mKate2+(red)) ESC colonies. DAPI (nuclei) is in blue. Representative images of four repeats. Scale bar: 100 µm. j, k Western blot analysis of mESC whole cell lysate with or without Cre recombination. j Total protein stain (red). k Western blot probed with streptavidin (cyan) or an anti-myc (red) antibody to detect myc-tagged BirA*G3. For each image, all lanes are from the same blot (see Source Data for uncropped blots). Representative results of three western blots. See also: Supplementary Fig. 11. Source data are provided as a Source Data file.
Fig 3: Identification of mouse teratoma-derived proteins in serum.a, b Teratoma and serum log2(BirA*G3-ER/wt) TMT ratios in three replicates. Each point is n = 3 comparisons, mean ± SEM log2TMT ratio. The points are colored by the enrichment score (E-S): the number of comparisons (from 9) in which TMT-ratio > threshold (score 9 is for most confident hits [red] and 0 is background [black]). c, d As the E-S increases, the fraction of proteins with putative signal peptides increases. Two-sided Fisher’s exact test. Teratoma p-values (****) from left to right (c): 5.11 · 10-198, 7.86 · 10-221, 1.03 · 10-232, 5.11 · 10-241, 2.05 · 10-230, 1.66 · 10-216, 4.73 · 10-194, 3.10 · 10-167. Serum p-values (****) from left to right (d): 1.00 · 10-30, 3.61 · 10-33, 1.82 · 10-35, 2.48 · 10-35, 1.23 · 10-36, 1.37 · 10-31, 1.63 · 10-24, 8.42 · 10-18. e, f BirA*G3-ER;Cre teratoma and serum hits were enriched for lower-abundance proteins. Protein abundance information was from an integrated entire organism PAX database for mouse67. Frequency vs log10 protein abundance plots. For representation, serum and teratoma data are shown as histograms with equal bin sizes with B-spline smooth fits (calculated using OriginPro 2020). Statistics were performed on original data. Kruskal–Wallis test and Benjamini, Krieger, Yekutieli Linear Two-Stage Step-Up FDR (two-tailed p-value). Teratoma p-values (****) from top to bottom (e): 0.000071, 0.000025, 0.000009, 6.72 · 10-12, 4.26 · 10-11, 4.26 · 10-11, 1.02 · 10-8, 6.79 · 10-7. Serum p-values (****) from top to bottom (f): 0.0004, 0.0005, 0.0005, 0.0005, 0.0005, 0.0009, 0.0041, 0.0204. g–i As the E-S increases, the fraction of serum hits that were identified in the teratoma increases (g–h colored as red in g). Proteins with increased scores are not enriched for proteins found in previous whole blood (cells removed) proteomics (i) (see “Methods” section). Two-sided Fisher’s exact test. p-values from left to right (h): 0.0044, 0.0002, 0.000025, 0.000005, 0.000012, 0.000005, 0.000001, 0.0001. p-values from left to right (i; N.S. means non-significant): 0.9161, 0.9136, 0.8243, 0.9093, >0.9999, 0.7987, 0.0850, 0.2001. See also: Supplementary Figs. 14 and 15a–n, Supplementary Table 9, Supplementary Data 4–6. Source data are provided as a Source Data file.
Fig 4: Analysis of biotin labeling of proteins in vivo in a mouse embryonic stem cell (mESC)–derived teratoma model.a Schematic representation of the teratoma study. GFP+ BirA*G3-ER mESC are shown in green, and mKate2+ BirA*G3-ER;Cre mESC are in red. b–c Hematoxylin and eosin staining of cryo-sectioned teratoma tissue. b', c' High magnification images. Representative results of three replicate images. Scale: 100 µm. d–o Analysis of reporter gene activity and BirA*G3 in cryo-sections of teratoma tissue. Scale bar: 50 µm. Green is GFP, red is mKate2, cyan is BirA, magenta is phalloidin, and white is DAPI. Representative images from 2 biological replicates with 2 sections per replicate. Another set of images was also acquired in testing the binding of antibodies. p–q Streptavidin labeling of biotinylated proteins from streptavidin pulldown of tumor (p) and serum samples (q). Note: the stronger signal in sample 2 in q reflects the significantly larger tumor mass in this individual. Representative results from six (p) and four (q) western blots. For each image, all lanes are from the same blot (see Source Data for uncropped western blots). See also: Supplementary Figs. 12 and 13. Source data are provided as a Source Data file.
Fig 5: Characterization of in vitro biotinylation in the BirA*G3–ER mouse embryonic stem cell (mESC) line.a Western analysis of streptavidin binding to biotinylated proteins following 24 h of labeling at the indicated concentrations of biotin. All lanes are from the same blot (see Source Data for uncropped blots). b Quantification of biotin labeling relative to whole protein in a. c Western analysis of streptavidin binding to biotinylated proteins labeled in 50 µM biotin for the indicated times. All lanes are from the same blot (see Source Data for uncropped blots). d Quantification of biotin labeling relative to total protein in c. In this figure, representative results are shown from two western blots each. Source data are provided as a Source Data file.
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