firefly renilla single tube kit  (Biotium)

Journal: bioRxiv

Article Title: Biomolecular condensates of Eya drive transcriptional co-activation associated with eye development

doi: 10.1101/2025.10.15.682020

Figure Lengend Snippet: A. Schematics of all truncated Eya constructs B-K. Representative image of S2 cell immunostained with lamin antibody to mark the nucleus and expressing (B) full length Eya, (C) Δ84 Eya, (D) Δ187 Eya, (E) Δ438 Eya, (F) Δ485 Eya, (G ) N-terminal 438 amino acid fragment of Eya, (H) ΔED Eya (485 Amino acid fragment from N terminal), (I) ΔPTP Eya (PST-TPM-PST domain deleted), (J) ΔTPM Eya (TPM region deleted), (K) PTP fragment of Eya (PST-TPM-PST). L. Schematic of the reporter gene-based transcription activation assay. S2 cells were transfected with the 8XARE-Firefly luciferase and Renilla luciferase to normalize the values along with Eya and So, Eya ΔPTP and So, Only Eya, only Eya ΔPTP, and only So. Transfected cells were later lysed and processed for dual luciferase assay using a luminometer. M. Quantitation of reporter gene activity normalized with Renilla luciferase activity shows that while the Eya and So can activate the reporter gene transcription, the ΔPTP mutant with So cannot activate the reporter gene transcription. The values for Eya+So were first normalized to 100 and were then used as a reference to normalize the other columns. RM one-way ANOVA with Geisser-Greenhouse correction was performed. The mean values of each column were compared with the mean of the Eya+So column with the help of Dunnett’s multiple comparison test. The p-values of every column compared to Eya +So were <0.0001. The experiment was repeated three times with 3 technical repeats for each time. Error bars represent SEMs. N. Representative image of S2 cell coexpressing Eya ΔPTP and So, showing no partitioning of So in condensates in the absence of Eya condensates.

Article Snippet: Cells were grown for 48 hours, followed by pelleting them down for the Luciferase assay using the Biotium Firefly & Renilla Luciferase Single Tube Assay Kit and taking the readings in a luminometer.

Techniques: Construct, Expressing, Activation Assay, Transfection, Luciferase, Quantitation Assay, Activity Assay, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Biomolecular condensates of Eya drive transcriptional co-activation associated with eye development

doi: 10.1101/2025.10.15.682020

Figure Lengend Snippet: A. Schematic showing alignment of the PST-TPM-PST region of Drosophila Eya with the corresponding region from Human Eya1. The residues inside the red boxes are residues associated with mutations in human patients. B. Quantitation of reporter gene activity normalized with Renilla luciferase activity for wild type and the three Eya mutants shows the G223S mutation to exhibit significantly reduced reporter gene activity. The values for Eya wt were normalized to 100. RM one-way ANOVA with Geisser-Greenhouse correction was performed, while the mean difference was calculated using Dunnett’s multiple comparison test, where Eya wt was considered as the reference point. The p-value for the G223S mutant was 0.0085. Error bars represent SEMs. C. Frames from live confocal imaging of S2 cells expressing wild-type Eya (top panel) showing fusion of the condensates (marked by arrow), whereas the Eya G223S mutant (bottom panel) condensates do not fuse. D. Frames from the FRAP experiment with wild-type Eya condensates (top panel) and G223S (bottom panel) showing the state of the recovery of the fluorescence after photobleaching (marked by a white box at the beginning and the end). E. FRAP recovery graph of wild-type Eya condensate and the G223S mutant. F. Quantitation shows G223S mutant condensates to have significantly reduced mobile fraction in comparison to the wild-type Eya. Unpaired Student’s t-test was done to compare the difference between mobile fractions. N for Eya wt =10 and for G223S =12. The p-value was <0.0001. Error bars represent SEMs.

Article Snippet: Cells were grown for 48 hours, followed by pelleting them down for the Luciferase assay using the Biotium Firefly & Renilla Luciferase Single Tube Assay Kit and taking the readings in a luminometer.

Techniques: Quantitation Assay, Activity Assay, Luciferase, Mutagenesis, Comparison, Imaging, Expressing, Fluorescence