Figure 9.
Figure 9. Compensatory regulation of actin isoforms in the somatodendritic and axonal compartments after depletion of Actα, Actβ, or Actγ. (A–C) RNA was prepared from actin knockdown and control motoneuron cultures and analyzed by quantitative RT-PCR. (A) mRNA levels of Actα are decreased after shRNA-mediated knockdown, whereas mRNA levels of Actβ and Actγ are not altered (**, P < 0.0037 for n = 5). (B) Depletion of Actβ leads to a 70% decrease in Actβ (***, P < 0.0005), a 5.5-fold increase in Actα (***, P < 0.0005), and a 2.6-fold increase in Actγ mRNA levels (*, P < 0.018 for n = 7). (C) Depletion of Actγ leads to a 80% decrease in Actγ (**, P < 0.001), a 1.6-fold increase in Actα, and a 2.6-fold increase in Actβ mRNA levels (**, P < 0.0054 for n = 5). (D–F) Quantitative RT-PCR analysis of RNA samples obtained from somatodendritic and axonal compartments of motoneuron cultures transduced with actin shRNA constructs. (D) In Actα-depleted motoneurons, mRNA levels of Actα are decreased in both somatodendritic and axonal compartments (**, P < 0.002), mRNA levels of Actβ are decreased in the somatodendritic compartment (* P < 0.047) and mRNA levels of both Actβ and Actγ are increased in the axonal compartment (*, P < 0.011 for n = 5). (E) In Actβ-depleted motoneurons, mRNA levels of Actβ are decreased in somatodendritic and axonal compartments (*, P < 0.014) and mRNA levels of Actα and Actγ are increased in both somatodendritic (*, P < 0.028) and axonal compartments (*, P < 0.014 for n = 4). (F) In Actγ-depleted motoneurons, mRNA levels of Actγ are decreased in somatodendritic (**, P < 0.002) and axonal compartments (**, P < 0.001) and mRNA levels of Actα are increased in somatodendritic (*, P < 0.046) and axonal compartments (**, P < 0.002 for n = 5). (G–I) Western blot analysis of cultured motoneuron lysates probed with specific Actα (G), Actβ (H), and Actγ (I) antibodies. (J) Quantification of blots in G showed 2.5-fold up-regulation of Actα protein after knockdown of Actβ (**, P < 0.004 for n = 4). (K) Quantification of blots in H showed 1.5-fold up-regulation of Actβ protein after knockdown of Actγ (***, P < 0.0003 for n = 8). (L) Quantification of blots in I showed 1.7-fold up-regulation of Actγ protein after knockdown of Actβ (**, P < 0.002 for n = 6). Statistical analysis was performed using a one-tailed Mann-Whitney test. Shown are mean ± SEM.

Compensatory regulation of actin isoforms in the somatodendritic and axonal compartments after depletion of Actα, Actβ, or Actγ. (A–C) RNA was prepared from actin knockdown and control motoneuron cultures and analyzed by quantitative RT-PCR. (A) mRNA levels of Actα are decreased after shRNA-mediated knockdown, whereas mRNA levels of Actβ and Actγ are not altered (**, P < 0.0037 for n = 5). (B) Depletion of Actβ leads to a 70% decrease in Actβ (***, P < 0.0005), a 5.5-fold increase in Actα (***, P < 0.0005), and a 2.6-fold increase in Actγ mRNA levels (*, P < 0.018 for n = 7). (C) Depletion of Actγ leads to a 80% decrease in Actγ (**, P < 0.001), a 1.6-fold increase in Actα, and a 2.6-fold increase in Actβ mRNA levels (**, P < 0.0054 for n = 5). (D–F) Quantitative RT-PCR analysis of RNA samples obtained from somatodendritic and axonal compartments of motoneuron cultures transduced with actin shRNA constructs. (D) In Actα-depleted motoneurons, mRNA levels of Actα are decreased in both somatodendritic and axonal compartments (**, P < 0.002), mRNA levels of Actβ are decreased in the somatodendritic compartment (* P < 0.047) and mRNA levels of both Actβ and Actγ are increased in the axonal compartment (*, P < 0.011 for n = 5). (E) In Actβ-depleted motoneurons, mRNA levels of Actβ are decreased in somatodendritic and axonal compartments (*, P < 0.014) and mRNA levels of Actα and Actγ are increased in both somatodendritic (*, P < 0.028) and axonal compartments (*, P < 0.014 for n = 4). (F) In Actγ-depleted motoneurons, mRNA levels of Actγ are decreased in somatodendritic (**, P < 0.002) and axonal compartments (**, P < 0.001) and mRNA levels of Actα are increased in somatodendritic (*, P < 0.046) and axonal compartments (**, P < 0.002 for n = 5). (G–I) Western blot analysis of cultured motoneuron lysates probed with specific Actα (G), Actβ (H), and Actγ (I) antibodies. (J) Quantification of blots in G showed 2.5-fold up-regulation of Actα protein after knockdown of Actβ (**, P < 0.004 for n = 4). (K) Quantification of blots in H showed 1.5-fold up-regulation of Actβ protein after knockdown of Actγ (***, P < 0.0003 for n = 8). (L) Quantification of blots in I showed 1.7-fold up-regulation of Actγ protein after knockdown of Actβ (**, P < 0.002 for n = 6). Statistical analysis was performed using a one-tailed Mann-Whitney test. Shown are mean ± SEM.

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