Journal: ACS Omega

Article Title: Dynamic Detection of the E3-PROTAC-Target Protein Ternary Complex In Vitro and In Vivo via Bimolecular Fluorescence Complementation

doi: 10.1021/acsomega.4c08186

Figure Lengend Snippet: Validation of the AKT-PROTAC-Reporter (APR) in U251 and 293T cells. (A) Left, the working principle of bimolecular fluorescence complementation (BiFC) technology based on EGFP; Right, a working diagram of a reporter dependent on PROTAC’s EGFP-based BiFC. (B) Schematic diagram of the plasmid NEGFP-AKT1-P2A-CEGFP-CRBN (EAEC). (C) HEK 293T cells with stable EAEC expression were treated with DMSO, MS1710 (1 μM), MS98 (2 μM), MS5033 (2 μM), or INY-03-041 (200 nM) for 12 h, and the intracellular green fluorescence was captured by fluorescence microscopy (20×). (D) The mean fluorescence intensity (MFI) at different time points after different PROTACs were used to treat U251 cells stably expressing EAEC. (E) The effects of DMSO and MS170 on stable overexpression of HA-EA and His-EC in U251 and 293T cells were studied. The protein immunoblotting results were representative of three independent replicates. After treatment with MS170, HA-EA could be significantly degraded, while His-EC showed no significant difference compared with control group. (F) Fluorescence microscopy (20×) was used to capture the change in the green fluorescence intensity of U251 and 293T cells with stable APR overexpression after treatment with DMSO and MS170 (1 μM) for 12 h. MS170 enables the APR to show microscopically captured green fluorescence inside cells. (G) U251 and 293T cells were digested and suspended, and then flow cytometry was used to further detect the change in the green fluorescence level after MS170 treatment. The proportion of fluorescent cells quantified in this experiment was based on the FITC channel signal intensity of 1 × 10 4 . ****: p < 0.0001, ***: p < 0.001, ns: not significant. Scale bars = 100 μm.

Article Snippet: After digestion of the vector with XbaI and Eco RI enzymes, the nucleotide sequences of the His tag, P2A, and HA tag were first synthesized on the treated vector by Sangon Biotech (Shanghai, China).

Techniques: Biomarker Discovery, Fluorescence, Plasmid Preparation, Expressing, Microscopy, Stable Transfection, Over Expression, Western Blot, Control, Flow Cytometry

Journal: eLife

Article Title: Visualizing the metazoan proliferation-quiescence decision in vivo

doi: 10.7554/eLife.63265

Figure Lengend Snippet:

Article Snippet: Sequenced-based reagent , NgoMIV-P2A(codon-de-optimized)-his-58-GFP-NheI , This study , Twist Biosciences , CATCCAAGCTCGGACATCGTGCCGGCGCGGGAAGTGGGGCCACGAACTTCAGTCTCCTCAAACAAGCCGGGGACGTCGAAGAGAACCCCGGGCCAATGCCACCAAAGCCATCTGCCAAGGGAGCCAAGAAGGCCGCCAAGACCGTCGTTGCCAAGCCAAAGGACGGAAAGAAGAGACGTCATGCCCGCAAGGAATCGTACTCCGTCTACATCTACCGTGTTCTCAAGCAAGTTCACCCAGACACCGGAGTCTCCTCCAAGGCCATGTCTATCATGAACTCCTTCGTCAACGATGTATTCGAACGCATCGCTTCGGAAGCTTCCCGTCTTGCTCATTACAACAAACGCTCAACGATCTCATCCCGCGAAATTCAAACCGCTGTCCGTTTGATTCTCCCAGGAGAACTTGCCAAGCACGCCGTGTCTGAGGGAACCAAGGCCGTCACCAAGTACACTTCCAGCAAGATGAGTAAAGGAGAAGAATTGTTCACTGGAGTTGTCCCAATCCTCGTCGAGCTCGACGGAGACGTCAACGGACACAAGTTCTCCGTCTCCGGAGAGGGAGAGGGAGACGCCACCTACGGAAAGCTCACCCTCAAGTTCATCTGCACCACCGGAAAGCTCCCAGTCCCATGGCCAACCCTCGTCACCACCTTCTGCTACGGAGTCCAATGCTTCTCCCGTTACCCAGACCACATGAAGCGTCACGACTTCTTCAAGTCCGCCATGCCAGAGGGATACGTCCAAGAGCGTACCATCTTCTTtAAGgtaagtttaaacatatatatactaactactgattatttaaattttcagGACGACGGAAACTACAAGACCCGTGCCGAGGTCAAGTTCGAGGGAGACACCCTCGTCAACCGTATCGAGCTCCAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGAATCGACTTCAAGGAGGACGGAAACATCCTCGGACACAAGCTCGAGTACAACTACAACTCCCACAACGTCTACATCATGGCCGACAAGCAAAAGAACGGAATCAAGGTCAACTTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagATCCGTCACAACATCGAGGACGGATCCGTCCAACTCGCCGACCACTACCAACAAAACACCCCAATCGGAGACGGACCAGTCCTCCTCCCAGACAACCACTACCTCTCCACCCAATCCGCCCTCTCCAAGGACCCAAACGAGAAGCGTGACCACATGGTCCTCCTCGAGTTCGTCACCGCCGCCGGAATCACCCACGGAATGGACGAGCTCTACAAGTCAGGAGCTAGCGGAGCCTACCCTTACGACG.

Techniques: Recombinant, Plasmid Preparation, Blocking Assay