Developmental Profiling of Tropomyosin Expression in Mouse Brain Reveals Tpm4.2 as the Major Post-synaptic Tropomyosin in the Mature Brain

Figures

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  FIGURE 1. Expression profile of tropomyosin (Tpm) isoforms in the brain throughout development and aging. Western blot analysis and quantification of E16.5, 1, 3, and 7-months-old mouse brain tissue for Tpm expression. (A) Tpm1.10 and Tpm1.12 expression, as detected by the 5–54 antibody. (B) Quantification of expression of Tpm1.10 and Tpm1.12 expression (∗) indicates significance for Tpm1.10, (#) indicates significance for Tpm1.12. (C) Expression of Tpm4.2 as detected by the δ9d antibody. (D) Quantification of (C). (E) Expression of Tpm3.1, 2, as detected by the γ9d antibody. (F) Quantification of (E). (mean ± SEM, n = 3, ∗/#P < 0.05, ∗∗/##P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

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  FIGURE 2. Expression profile of tropomyosin (Tpm) isoforms in the brain throughout development and aging. Western blot analysis and quantification of E16.5, 1, 3, and 7-months old-mouse brain tissue for Tpm expression. (A) Tpm3.3, 5, 8, 9 expression as detected by the γ/9a antibody. (B) Quantification of (A). (C) Expression of Tpm3.4,7, as detected by the γ9c antibody. (D) Quantification of (C). (mean ± SEM, n = 3, ∗P < 0.05, ∗∗P < 0.01).

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  FIGURE 3. Expression profile of tropomyosin (Tpm) isoforms Tpm1.10 and Tpm1.12 (utilizing the 5–54 and Tm311 antibodies) in the brain homogenate (BH) and post-synaptic density (PSD) of wild type mouse brains. Western blot of 1-month (A), 3-months (B), and 7-months (C) brain homogenate and post-synaptic densities show localization of Tpm1.12 in the brain homogenate, but not the post-synaptic compartment throughout the age groups. Total actin and Ponceau staining of blots demonstrates equal loading. (D) Western blot analysis of the immunoreactivity for Tpm1.10 and Tpm1.12 in the brain homogenate, synaptic junction and post-synaptic compartment of 3-months-old wild-type mice. (E) Due to the poor Tpm1.10 signal in the synaptic junction, the membrane was overexposed to detect Tpm1.10 expression. (F) To confirm the specificity of the Tpm1.10 signal in (E) the Tm311 antibody, which detects Tpm1.10 but not Tpm1.12 was used, indicating immunoreactivity for Tpm1.10 in the synaptic junction.

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  FIGURE 4. Western blot expression profile of tropomyosin (Tpm) isoform Tpm4.2 (utilizing the δ9d antibody) in the brain homogenate (BH) and post-synaptic density (PSD) of wild type mouse brains. (A) Western blot analysis of 1-month-old brain homogenate and PSD fractions. (B) Quantification of (A) normalized to expression in the brain homogenate. (C) Western blots of 3-months-old brain homogenate and PSD fractions. (D) Quantification of (C) normalized to expression in the brain homogenate. (E) Western blot of 7-months-old brain homogenate and PSD fractions. (F) Quantification of (E) normalized to expression in the brain homogenate. Total actin and Ponceau staining are used as loading controls. (mean ± SEM, n = 4, ∗∗P < 0.01, ∗∗∗∗P < 0.0001).

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  FIGURE 5. Western blot analysis of expression profile of Tpm3 exon 9a-containing tropomyosin (Tpm) isoforms (utilizing the γ9a mouse monoclonal antibody) in the brain homogenate (BH) and post-synaptic density (PSD) of wild type mouse brains. (A) Western blot of 1-month-old brain homogenate and PSD fractions. (B) Quantification of (A) normalized to expression in the brain homogenate. (C) Western blot of 3-months-old brain homogenate and PSD fractions. (D) Quantification of (C) normalized to expression in the brain homogenate. (E) Western blot of 7-months-old brain homogenate and PSD fractions. (F) Quantification of (E) normalized to expression in the brain homogenate. Total actin and Ponceau staining are used as loading controls. (mean ± SEM, n = 4, ∗P < 0.05, ∗∗∗∗P < 0.0001).

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  FIGURE 6. Expression profile of Tpm3 exon 9c-containing tropomyosin (Tpm) isoforms utilizing the γ9c sheep polyclonal antibody in the brain homogenate (BH) and post-synaptic density (PSD) of wild type mouse brains. (A) Western blot of 1-month-old brain homogenate and PSD fractions. (B) Quantification of (A) normalized to expression in the brain homogenate. (C) Western blot of 3-months-old brain homogenate and PSD fractions. (D) Quantification of (C) normalized to expression in the brain homogenate. (E) Western blot of 7-months-old brain homogenate and PSD fractions. (F) Quantification of (E) normalized to expression in the brain homogenate. Total actin and Ponceau staining are used as loading controls. (mean ± SEM, n = 4, ∗P < 0.05).

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  FIGURE 7. Western blot analysis of expression profile of Tpm3.1 and Tpm3.2 utilizing the polyclonal γ9d sheep antibody in the brain homogenate and post-synaptic density (PSD) of wild type mouse brains. (A) Western blots of 1-month-old brain homogenate and PSD fractions. (B) Quantification of (A) normalized to expression in the brain homogenate. (C) Western blots of 3-months-old old brain homogenate and PSD fractions. (D) Quantification of (C) normalized to expression in the brain homogenate. (E) Western blot of 7-months-old brain homogenate and PSD fractions. (F) Quantification of (E) normalized to expression in the brain homogenate. Total actin and Ponceau staining show equal loading. (mean ± SEM, n = 4, ∗∗P < 0.01, ∗∗∗P < 0.001).

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  FIGURE 8. Western blot analysis of the relative expression of tropomyosin (Tpm) isoforms at the post-synaptic density (PSD) in 1, 3, and 7-months-old mice. (A) Expression profile of Tpm3 exon 9a-containing Tpm isoforms utilizing the γ9a mouse monoclonal antibody in the PSD of 1, 3, and 7-months-old mice. (B) Quantification of (A) normalized to expression at 1-month of age. (C) Expression profile of Tpm3.1 and Tpm3.2 utilizing the polyclonal γ/9d sheep antibody in the PSD of 1, 3, and 7-months-old mice. (D) Quantification of (C) normalized to expression at 1-month of age. (E) Expression profile of Tpm4.2, utilizing the δ9d antibody, in the PSD of 1, 3 and 7-months-old mice. (F) Quantification of (E) normalized to expression at 1-month of age. (mean ± SEM, n = 4, ∗P < 0.05, ∗∗P < 0.01).

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  FIGURE 9. Total protein expression of Tpm3 and Tpm4 products. (A) Western blot analysis of absolute levels of Tpm expression from Tpm3 and Tpm4 in the brain homogenate of 1-month-old mice. Note the higher molecular weight of recombinant Tpm constructs due to a His-tag. (B) Quantification of absolute levels of Tpm isoforms from Tpm3 and Tpm4 in the brain homogenate of mice. (C) Quantification of absolute levels of Tpm isoforms from Tpm3 and Tpm4 in the post-synaptic density of mice. (D) Comparative levels of the contribution of Tpm3 versus Tpm4 isoforms to the total output from Tpm3 and Tpm4 in brain homogenate (BH) and the post-synaptic density (PSD) fraction. (E) Fold difference in expression of Tpm4.2 compared to Tpm3 isoforms in brain homogenate. (I) Fold difference in expression of Tpm4.2 compared to Tpm3 isoforms in the post-synaptic density fraction. Shown are p-values comparing individual Tpm isoforms. (mean ± SEM, n = 4). Significant differences in (E) are highlighted in blue.