recombinant egfp  (Cambridge Bioscience)

Journal: bioRxiv

Article Title: Amplicon/Protein Bead Display enables quantitative in vitro biochemistry at scale

doi: 10.64898/2026.05.28.728566

Figure Lengend Snippet: a . Schematic of DNA templates encoding expression of a 25-variant FLAG library expressed as SNAP and eGFP fusion proteins on APBs. b . Schematic of experiments to detect binding between bead-displayed FLAG epitopes and M2 anti-FLAG antibodies using a Cy5-labeled secondary antibody. c . Representative brightfield and fluorescence images of APBs after incubation with M2 and Cy5-labeled secondary antibodies reporting on per-bead expression levels (GFP intensities), and antibody binding (Cy5 intensities). d . Box plots of per-bead median Cy5 pixel intensities (arbitrary units) for blank and GFP-positive beads. e . Schematic of single-bead sorting and sequencing workflow for validating genotype-phenotype linkages. FACS plot (far left) displays measured GFP (x-axis) and Cy5 (y-axis) intensities for the 384 GFP-positive beads sorted into a multi-well plate. f . Bar plots of expected (based on Poisson loading at λ = 0.1) and observed fractions of single-variant beads in the GFP-positive library. Error bars represent the Poisson-derived standard deviation. g . Measured Cy5 versus eGFP fluorescence intensities for all beads displaying three representative FLAG variants. Circles indicate single-variant beads; X’s indicate multi-variant beads. Red dashed lines indicate median Cy5/GFP ratio; gray shading shows interquartile range [0.25,0.75]. h . Median Cy5/GFP ratios calculated from all beads bearing a given variant versus single-variant beads only. Error bars denote interquartile range [0.25, 0.75]. i . Coefficient of variation (CV%) of median Cy5/GFP ratios across 1,000 bootstrap samples as a function of beads sampled per variant. j . Sequence logo showing relative position-normalized Cy5/GFP ratios for single amino acid substitutions (A, L, or E) at each position in the FLAG epitope. Red letters indicate wildtype residues; grey letters indicate substitutions.

Article Snippet: A fluorescence standard curve for estimating protein concentration on beads was generated using vortexed emulsion droplets containing known concentrations of recombinant eGFP protein (Origene).

Techniques: Expressing, Variant Assay, Binding Assay, Labeling, Fluorescence, Incubation, Sequencing, Derivative Assay, Standard Deviation, FLAG-tag

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Levels of the 7TGP fluorescent Wnt reporter in HEK293T cells transfected with control siRNA, Dvl1-specific siRNA, Nup358-specific siRNA, or a combination of Nup358-specific siRNA and Dvl1-specific siRNA and treated with either Wnt3a or vehicle. b. Western blot analysis and relative quantification of protein levels of Dvl1 and Axin1 in HEK293T cells transfected with either control or Nup358-specific siRNA. α-Tubulin and GAPDH were used as loading controls. Data are expressed as mean ± SD. **p ≤ 0.01. c. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with either control or Nup358-specific siRNA. d. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with control or Nup358-specific siRNA and treated with vehicle or 1,6-hexanediol 5% for 2 minutes. e. Fluorescence recovery after photobleaching (FRAP) and fusion analysis of Dvl1 condensates in HEK293T cells transfected with Nup358-specific siRNA and EGFP-Dvl1.

Article Snippet: Reporter activity was validated by treating the stable line with recombinant Wnt3a (100 ng/mL, Proteintech HZ-1296) and confirming EGFP induction via fluorescence microscopy.

Techniques: Transfection, Control, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Schematic representing different functional domains of Nup358. b. Fluorescent Wnt reporter activity in HEK293T cells transfected with control siRNA, Nup358-specific siRNA, Ubc9-specific siRNA, or treated with the SUMOylation inhibitor 2-D08. c. Schematic representing different HA-tagged truncated forms of Nup358 that were transiently expressed in HEK293T cells. d. Fluorescent Wnt reporter activity in HEK293T cells transfected with either control or Nup358-specific siRNA alone or in combination with different HA-tagged truncated forms of Nup358 were analyzed by confocal microscopy (top) and quantified (bottom). Data are expressed as mean ± SD. ****p ≤ 0.0001 e. Western blot analysis of co-immunoprecipitation of HA-Nup358 and EGFP-Dvl1 transiently expressed in HEK293T cells. GAPDH was used as loading control. f. Immunofluorescence of HEK293T cells transfected with HA-Tagged Nup358 1–1133 and EGFP-Dvl1and stained with an anti-HA antibody. g. Immunofluorescence staining for endogenous Dvl1 and Nup358 in HEK293T cells transfected with either scramble control or Nup358-specific siRNA alone or in combination with HA-Tagged Nup358 1–1133 fragment were analyzed by confocal microscopy (top) and quantified (bottom). Data are expressed as mean ± SD. **p ≤ 0.01.

Article Snippet: Reporter activity was validated by treating the stable line with recombinant Wnt3a (100 ng/mL, Proteintech HZ-1296) and confirming EGFP induction via fluorescence microscopy.

Techniques: Functional Assay, Activity Assay, Transfection, Control, Confocal Microscopy, Western Blot, Immunoprecipitation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: C9ORF72-derived polyGR polypeptides disrupt passive nucleocytoplasmic transport by tuning protein affinity for the nuclear pore barrier

doi: 10.64898/2026.03.16.711670

Figure Lengend Snippet: a,b, Passive nuclear import assays in permeabilized HeLa cells reveal that 10 µM GR 20 peptide markedly increases nuclear accumulation of 20 kDa fluorescent dextran compared with untreated (UT), GP 20 , or PA 20 – representative images and mean cellular partition coefficient (nucleoplasm to background intensity ratio). Stats: one-way ANOVA with Tukey’s multiple comparisons test; n= 7 biological replicates for untreated, 3 for polyGR, 2 for polyGP and polyPA. c,d, GR 20 selectively enhances nuclear import of EGFP but not mCherry (experiments performed separately but shown on same graph for ease of comparison). Stats: two-tailed paired t -test; n= 3 biological replicates. e, Recombinantly purified human Nup98 FG domain forms condensates that model NPC permeability; inert clients mCherry and EGFP are excluded from the Nup98 FG phase, whereas the transport receptor NTF2 readily partitions into the Nup98 FG phase. f, Fluorescently (Alexa488)-labelled GR 20 , but not PA 20 , partitions into Nup98 FG condensates, demonstrating direct and specific engagement with the FG network. g,h , Consistent with cellular data, GR 20 increases EGFP partitioning into Nup98 FG condensates compared to untreated, whereas GP 20 and PA 20 have no effect; images show representative condensate intensity with heatmap scaling, together with corresponding mean condensate partition coefficients (PC) (inside to outside particle intensity ratio). Stats: one-way ANOVA with Tukey’s multiple comparisons test; n = 5 biological replicates, ≥5 condensates per replicate. i,j Consistent with cellular data, GR 20 does not alter mCherry partitioning into Nup98 FG condensates relative to untreated unlike EGFP (replotted from for ease of comparison); images show representative condensate intensity with heatmap scaling, together with corresponding mean condensate partition coefficients. Stats: two-tailed paired t -test: n= 5 biological replicates for EGFP, 3 for mCherry, ≥5 condensates per replicate. Data are shown as mean ± SEM. **** p < 0.0001; ** p <0.01; * p <0.05; ns, not significant. Scale bars = 20 µm for cells, 2 µm for condensates.

Article Snippet: Recombinant EGFP was obtained from Cambridge Bioscience (STA-201) and mCherry from Abcam (ab199750).

Techniques: Comparison, Two Tailed Test, Purification, Permeability