Kalirin9 binds p75. A, the recombinant Kalirin9 and 12 bind
the cytoplasmic domain of p75 in vitro. GST or GST-p75ICD
was incubated with the lysates from 293T cells that were transfected with the
Myc-tagged wild type Kalirin9 or 12. The bound Kalirins were detected by Myc
Western analysis (W). For input, 5% of the lysates that were used for
GST pulldown were loaded as controls. B, Kalirin is expressed among
p75+ Purkinje neurons in the P5 cerebellum. P5 rat sagittal
sections were stained with an anti-p75 antibody, 192, and pan-Kalirin
antibodies (28). The
arrows point to doubly stained Purkinje neurons. Scale bar,
12.5 μm. C, Kalirin9, and not Kalirin12, associates with p75 and
NgR in the developing cerebellum. Upper panel, the cerebellar lysates
from P5 rat pups were subjected to immunoprecipitation (IP) with a
p75 antibody and a control IgG. The bound proteins were simultaneously probed
with pan-Kalirin and NgR antibodies using a divided membrane. For detection of
p75, the same membrane was stripped and reprobed. *, nonspecific
bands. Lower panel, detection of Kalirin9 and p75 in reciprocal
immunoprecipitation with NgR antibody. The bound proteins were simultaneously
probed with pan-Kalirin and NgR antibodies using a divided membrane. As a
control, the same blot was reprobed for NgR following membrane stripping.
*, a nonspecific band; ○, IgG heavy chain. D, input
controls for C. Note that Kalirin9 is the major isoform present in
CGN cultures, whereas Kalirin12 is not expressed at detectable level.