Journal: Andrology
Article Title: Identification of MEF2A, MEF2C, and MEF2D interactomes in basal and Fsk-stimulated mouse MA-10 Leydig cells.
doi: 10.1111/andr.70051
Figure Lengend Snippet: FIGURE 1 Characterization of MEF2‒TurboID constructs and biotinylation analysis. (A) Schematic representation of the TurboID constructs, each with a 3xHA tag and a nuclear localization signal (NLS). MEF2‒TurboID fusions have MEF2 positioned at either the N- or C-terminal region of TurboID. TurboID‒GFP and empty TurboID were used as controls. (B) Western blot of 10 µg of nuclear extracts from MA-10 Leydig cells transfected with MEF2A-, MEF2C-, or MEF2D‒TurboID constructs or empty expression vector (Ctr). HA-tag detection confirmed the expression of MEF2A and MEF2D fused to the C-terminal of TurboID (C-MEF2A and C-MEF2D, 100 kDa), and MEF2C to the N-terminal (N-MEF2C, 85 kDa). LaminB was used as a loading control. (C) Reporter assay in MA-10 Leydig cells (left) and CV-1 fibroblast cells (right). Cells were co-transfected with 3xMEF2-luc reporter (pcDNA Ctr), wild-type (WT) MEF2 factors, or MEF2‒TurboID factors (N or C), with or without ERK5 (+ or −). Asterisks (*) indicate a significant difference (p < 0.05) between ERK5 alone (control, white hashed bar) and ERK5 + MEF2 constructs (MEF2‒TurboID, either N- or C-terminal, and WT, colorful hashed bars). Hashtags (#) indicate a significant difference (p < 0.05) between MEF2 alone (colorful solid bar) and ERK5 + MEF2 constructs (colorful hashed bars). Results shown are representative of four independent experiments. (D) Western blot analysis of 30 µg of total protein extracts from MA-10 Leydig cells transfected with C-MEF2A‒TurboID (C-terminal), and either left untreated (−) or treated (+) with 10 µM of forskolin (Fsk) and/or 50 µM of biotin for 4 h. GAPDH was used as a loading control. (E) Western blot of biotinylated proteins in MA-10 Leydig cells transfected with C-MEF2A‒TurboID, TurboID‒MEF2C-N, and C-MEF2D‒TurboID. Blot includes an input 4 h sample (before streptavidin bead enrichment) and biotinylated proteins eluted from streptavidin beads after 1 or 4 h of biotin supplementation, as well as a no-biotin control (−). Streptavidin‒HRP was used to visualize biotinylation.
Article Snippet: Expression vectors for ERK5 (pcDNA3-HA-ERK5) (Addgene plasmid #65244, http://n2t.net/addgene:65244, RRID:Addgene_65244) and the constitutively active MEK5 (pcDNA3-MEK5DD-HA) (Addgene plasmid #65247, http://n2t.net/addgene:65247, RRID:Addgene_65247) were gifts from Dr. Axel Ullrich.22 The 3xHATurboID-NLS_pcDNA3 was a gift from Alice Ting (Addgene plasmid #107171, http://n2t.net/addgene:107171, RRID:Addgene_107171).
Techniques: Construct, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, Reporter Assay
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