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. 2020 Jul 17;14:42. doi: 10.3389/fnana.2020.00042

FIGURE 1.

FIGURE 1

Validation of Tcf4-LGSL mice that faithfully report TCF4 expression. (A) Schematic of the strategy to generate C57BL/6J mice carrying the LoxP-P2A-GFP-STOP-LoxP cassette upstream of the basic helix-loop- helix region. Adenovirus splicing acceptor is shown by the blue box. (B) Representative Western blot for TCF4, GFP, and GAPDH loading control protein in embryonic brain lysates from Tcf4+/+ (WT), Tcf4LGSL/+ (Het), and Tcf4LGSL/LGSL (Homo) mice. The TCF4 antibody (recognizes mouse TCF4 aa 50–150) is designed to detect a long isoform of TCF4. We detected a TCF4 full length protein (TCF4-FL) band at approximately 76 kDa that corresponds to the long isoform in WT lysates. A TCF4 truncated protein (TCF4-Trunc.) was detected at approximately 65 kDa in Het lysates. A band for GFP or GAPDH protein was detected at approximately 26 or 35 kDa, respectively. (C) Quantification of Western blotting for TCF4-FL and for GFP. (D–F) Dual fluorescence ISH for Tcf4 (magenta) and GFP (green) from PFC of P80 WT and Tcf4LGSL/+, and CA1 of Tcf4LGSL/+ mice. Asterisk indicates a cell expressing only Tcf4, and arrows indicate cells co-expressing Tcf4 and GFP. Insets are higher magnifications. Scale bars = 10 μm. (G,H) Quantification of GFP-positive and -negative cells in Tcf4-expressing cells in the PFC and CA1 region (n = 3 mice). Data represent mean ± SEM.