Journal: Communications Biology

Article Title: POT, an optogenetics-based endogenous protein degradation system

doi: 10.1038/s42003-025-07919-x

Figure Lengend Snippet: a Schematic of the POT system. An N-terminal peptide was used to target the specific protein of interests. Blue light illumination promotes the CRY2olig oligomerization, which induces the TRIM21-mediated protein degradation. Of note, the protein icons used in the figure are symbolic only and do not represent the actual size of the proteins. b , c COS-7 cells expressing POT or control plasmid (POTΔTRIM) were stimulated with different intensities of 488 nm laser (10 s every 1 min). The cells were imaged by confocal microscope ( b ) and mCherry fluorescence was quantified ( c ). Cells were stimulated with different intensity of 488 nm laser (10 s every 1 min). n = 29, 18, 33, 18 cells for groups in POTΔTRIM, 0.5 mW/cm 2 , 1 mW/cm 2 and 2 mW/cm 2 , respectively. Scale bars: 10 μm. d Immunofluorescence images of COS-7 cells expressing POT-PI3K (red), before and 2 hours after illumination with blue light LED array (470 ± 10 nm; 1 mW/cm 2 , 1 s every 6 s). Cells were fixed and immune-stained with antibodies against PI3K 110α (green). (R)-MG132 was used to abolish protein degradation during immunofluorescence. Scale bars, 10 μm for full cell images and 1 μm for magnified images. e Intensity profile of PI3K 110α and POT-PI3K along the white lines on magnified images in ( d ). f Pearson’s correlation coefficient for POT-PI3K with PI3K 110α ( n = 17 cells). All data were presented as mean ± SEM. Statistical significance is based on two-tailed Student’s t -test and is represented with labels, ns (P > 0.05), **** ( P ≤ 0.0001).

Article Snippet: Following this, iSH2 (from Addgene #66839), mCherry (from Addgene #66839), and CRY2olig (CRY2 E490G mutant synthesized by Genewiz) were sequentially inserted into the EcoR1 site, with flexible (GS) linkers and specific restriction enzyme cutting sites placed between each element to facilitate cloning.

Techniques: Expressing, Control, Plasmid Preparation, Microscopy, Fluorescence, Immunofluorescence, Staining, Two Tailed Test

Journal: PLOS ONE

Article Title: Bioinformatics and In silico approaches to identify novel biomarkers and key pathways for cancers that are linked to the progression of female infertility: A comprehensive approach for drug discovery

doi: 10.1371/journal.pone.0265746

Figure Lengend Snippet: Role of some functionally essential mutated amino acids in the iSH2 domain.

Article Snippet: The domain part of PIK3R1 (chain B) protein is inter-Src homology-2 (iSH2) provided 400 to 600 amino acid sequence identified through Interpro server [ , ].

Techniques: Mutagenesis, Activity Assay, Positive Control, Transformation Assay

Journal: Viruses

Article Title: Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

doi: 10.3390/v12030338

Figure Lengend Snippet: Interaction between 1918 NS1 and p85β (PDB ID: 6U28). ( A ) Crystal structure of the 1918 NS1 ED-CTT :p85β iSH2 complex. The position of residue 187 is shown as a blue sphere. Biolayer interferometry (BLI)-derived binding isotherms of ( B ) 1918 NS1E D-CTT and CRK-II, ( C ) 1918 NS1 ED-CTT and CRK-L. Insets: representative BLI sensorgrams with different analyte concentrations are shown by different colors. K D values and uncertainties are the global fitting result of three repeated data. ( D ) BLI sensorgram of 1918 NS1 ED-ΔCTT binding to CRK-L.

Article Snippet: Genes encoding 1918 NS1 ED-CTT (residues 86–230), human p85β iSH2 (residues 435–599), human CRK-II (residues 1–304) and CRK-L (residues 1–303) proteins were prepared by gene-synthesis service from Genscript (Piscataway, NJ, USA).

Techniques: Residue, Derivative Assay, Binding Assay

Journal: Viruses

Article Title: Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

doi: 10.3390/v12030338

Figure Lengend Snippet: Ternary interaction between 1918 NS1, p85β, and CRK. ( A ) Schematic showing the NS1-bridged (left) and p85β-bridged (right) ternary complex. BLI-derived binding isotherms between the 1918 NS1 ED-CTT :p85β complex and ( B ) CRK-II and ( C ) CRK-L. Insets: representative BLI sensorgrams with different analyte concentrations are shown by different colors. K D values and uncertainties are the global fitting result of three repeated data.

Article Snippet: Genes encoding 1918 NS1 ED-CTT (residues 86–230), human p85β iSH2 (residues 435–599), human CRK-II (residues 1–304) and CRK-L (residues 1–303) proteins were prepared by gene-synthesis service from Genscript (Piscataway, NJ, USA).

Techniques: Derivative Assay, Binding Assay

Journal: Viruses

Article Title: Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

doi: 10.3390/v12030338

Figure Lengend Snippet: The C-terminal tail (CTT) of 1918 NS1 mediates the interaction with CRK. ( A ) Crystal structure of the 1918 NS1 ED-CTT :p85β iSH2 complex. The region with missing electron densities are shown as a dashed black line. The PRM region is marked by a blue circle. ( B ) Representation of crystallographic B-factors of 1918 NS1 ED-CTT in complex with p85β. BLI-derived binding isotherms between p85β iSH2 and ( C ) 1918 NS1 ED-ΔCTT and ( D ) CRK-II. The result of p85β iSH2 and CRK-II was not fit because the affinity was too weak to fit reliably. Insets: representative BLI sensorgrams with different analyte concentrations shown by different colors. K D values and uncertainties are the global fitting result of three repeated data.

Article Snippet: Genes encoding 1918 NS1 ED-CTT (residues 86–230), human p85β iSH2 (residues 435–599), human CRK-II (residues 1–304) and CRK-L (residues 1–303) proteins were prepared by gene-synthesis service from Genscript (Piscataway, NJ, USA).

Techniques: Derivative Assay, Binding Assay

Journal: Viruses

Article Title: Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

doi: 10.3390/v12030338

Figure Lengend Snippet: Fuzzy electrostatic interaction between CRK and the 1918 NS1:p85β complex. ( A ) Crystal structure of the free nSH3 (left) of CRK-II and its complex (right) with PRM of 1918 NS1 (PDB ID: 5UL6). The protein surface is colored according to electric potential at neutral pH from -5 kT (red) to +5 kT (blue). (right panel) Positively charged residues of PRM are shown in blue. BLI-derived binding isotherms between the 1918 NS1 ED-CTT :p85β iSH2 complex and ( B ) CRK-II and ( C ) CRK-L in the presence of 1M NaCl. ( D ) BLI-derived binding isotherm between 1918 NS1 ED-CTT and p85β iSH2 in the presence of 1M NaCl. Insets: representative BLI sensorgrams with different analyte concentrations are shown by different colors. ( E ) A schematic model showing how 1918 NS1 might enhance activation of PI3K using hijacked CRK.

Article Snippet: Genes encoding 1918 NS1 ED-CTT (residues 86–230), human p85β iSH2 (residues 435–599), human CRK-II (residues 1–304) and CRK-L (residues 1–303) proteins were prepared by gene-synthesis service from Genscript (Piscataway, NJ, USA).

Techniques: Derivative Assay, Binding Assay, Activation Assay