Fig. 7
Verification of ɑSMA antibody specificity with ACTA2 gene silencing method and assessment of ɑSMA protein levels in human dentate gyrus relative to control regions. (A) Western blot detection of αSMA, β-tubulin, F-actin and microtubule associated protein 2 (MAP2) in lysates from a representative set of cultured SY-SH5Y cells in the untreated (blank) (−/−), ACTA2 siRNA treated (+/−) and negative control siRNA treated (−/+) groups. The αSMA signal is greatly reduced in the ACTA2 siRNA treated cells, whereas that of the β-tubulin, F-actin and MAP2 proteins are comparable between the cell groups. (B) Quantitation from 3 separate culture experiments. (C) Microdissection of the dentate gyrus (DG), temporal neocortical (TC), frontal neocortical (FC) and basal artery (BA) samples from postmortem human brain. (D) Representative membrane images showing the optimization of the loading amounts of human frontal cortical and BA extracts to blot αSMA protein. (E, F) Representative immunoblot images showing the detection of αSMA relative to reference proteins in the regional brain and BA samples., with the amount of sample loading indicated. Note the increased signal in DG relative to FC, TC and cerebellar cortical (CBL) lysates (E, G), and similar levels of collagen IV and neuron-specific nuclear antigen (NeuN) in these regional samples (F). (H, I) Immunoblot images and quantification showing a higher αSMA protein level in the DG than TC lysates from an additional set of brains (n = 4) blotted with the rabbit and goat αSMA antibodies, respectively. Also note that the levels of GAPDH are reduced or become undetectable in the BA extracts with the dilution of sample loading (D, E, F, H). *: Significantly different per posthoc test.
