Generation of <i>HSPB2</i> cardiac specific overexpressors and effects on sHSP expression in transgenic mice (HSPB2cTg).

<p><b>(A)</b> A schematic diagram illustrates the human heat shock protein B2 (HSPB2) under the control of the α-myosin heavy chain (α-MyHC) promoter: the 3 black boxes in the promoter represent noncoding exons in the 5’-untranslated region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133994#pone.0133994.ref010" target="_blank">10</a>] and polyA splicing is provided by the human growth hormone sequences downstream of the inserted cDNA (hGH). <b>(B)</b> Representative Western blots of cardiac samples of transgenic (HSPB2cTg) mice and non-transgenic (HSPB2NTg) littermates (n = 4 animals per group) prepared as detergent soluble (supernatant) and non-soluble (pellet) portions, respectively. Protein levels of HSPB2 expression is markedly elevated (~4 x fold) in the HSPB2cTg hearts compared with non-transgenic (HSPB2NTg) littermates when assessed by western blot using anti-HspB2 antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133994#pone.0133994.ref009" target="_blank">9</a>]. Representative small heat shock proteins (sHSPs—HspB1, HspB3, HspB5, HspB6 and HspB8) exhibit similar levels in detergent soluble extracts, while HspB6 is significantly elevated in detergent insoluble fractions. Protein concentration was determined and western blots were performed using anti-sHSP (HspB1, HspB2, HspB5, HspB6 and HspB8) with anti-histone3 and anti-GAPDH as loading controls as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133994#pone.0133994.ref009" target="_blank">9</a>]. In addition, anti-HSPB3 antibodies were obtained from StressMarq Biosciences Inc and HspB3 levels were determined separately, which is indicated by a black line. <b>(C)</b> Densitometry-based quantification of the levels of protein in the immunoblots in Fig 1B. * <i>P</i><0.05 when compared with corresponding HSPB2NTg group.