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. 2007 Sep 12;27(37):10037–10046. doi: 10.1523/JNEUROSCI.0361-07.2007

Figure 2.

Figure 2.

PIASx is expressed in the cerebellar cortex. A, PIASx mRNA is expressed in cerebellar granule neurons. RNA was extracted from HEK293T cells (293T) transfected with the PIASx expression plasmid or control plasmid as well as primary granule neurons from P6 rat pups cultured for 5 d (P6 plus 5 DIV) and subjected to RT-PCR with primers specific for PIASx or GAPDH as an internal control. B, PIASx protein is expressed in cerebellar granule neurons. Lysates of granule neurons (P6 plus 5 DIV; 30 μg of lysate), HEK293T cells transfected with the PIASx expression plasmid (5 μg of lysate), were immunoblotted with a PIASx antibody. The asterisk indicates a nonspecific band in HEK293T cell lysates. In granule neurons, the PIASx antibody recognizes two distinct isoforms of PIASx (PIASxα and the larger PIASxβ) indicated by two arrowheads. In HEK293T cells, the antibody recognizes overexpressed PIASxα and several bands that run with much higher mobility that represent posttranslationally modified PIASxα. C, PIASx is expressed in the cerebellar cortex. Left, Immunostaining of cryosectioned P8 cerebella with a rabbit antibody to PIASx (Moilanen et al., 1999) reveals PIASx immunoreactivity in cells within the IGL (arrowhead) and in Purkinje cells (arrow). Right, Nuclear stain with the DNA dye bisbenzimide (Hoechst) showing layers of the cerebellar cortex. Scale bar, 100 μm. D, PIASx is expressed in Purkinje neurons and IGL neurons. Immunohistochemistry of P8 cerebella with the PIASx antibody (I, IV, VI) as in C together with a mouse monoclonal antibody to calbindin (II) or the neuron-specific tubulin type III (Tuj1) (V, VI). Hoechst is shown in III. The top panels (I–III) show that PIASx is expressed in calbindin-positive Purkinje neurons. Arrowheads in VI indicate a few examples of IGL granule neurons double-labeled with Tuj1 and PIASx. Scale bar: top, 100 μm; bottom, 50 μm. E, PIASx is expressed in the rat cerebellum in the first and second weeks postnatally. Lysates of the cerebellum of animals with the indicated age were immunoblotted with the PIASx or the ERK1/2 antibody, the latter serving as control for loading. Ctrl, Control; EGL, external granule layer; 293T, HEK293T.