Figure 4: Biochemical analysis of binding partners of CDC42EP4 in Cdc42ep4fl/fl and Cdc42ep4−/− cerebella.
From: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance
(a) Co-IP/IB assay of CDC42EP4 with representative septin subunits and GLAST from WT and KO cerebellar lysates. (Input) IB for SEPT4, SEPT7, SEPT2 and GLAST, respectively, detected a quadruplet of 54, 52, 48 and 44 kDa, a doublet of 51 and 48 kDa, a single 42 kDa band and a broad 55 kDa band in the cerebellar lysate. (IP) Anti-CDC42EP4 antibody pulled down SEPT4, SEPT7, SEPT2 and GLAST only from WT cerebellar lysate. The graphs show densitometric quantification of the yield (n=3, ***P<0.001, **P<0.01, *P<0.05, NS, P>0.05 by one-way ANOVA with post hoc Tukey test). (Note: the extraction condition including the lysis buffer composition was optimized to detect GLAST, which was distinct from the one used mainly for the proteomic analysis (Table 1). See Methods.) (b–d) Pellet/supernatant assay results on the quantity and extractability of SEPT7, SEPT4 and GLAST in WT and KO cerebella. There was no significant difference in their amount and partitioning by genotype (n=3, NS, P>0.05 by t-test). The same membranes were reprobed for α-tubulin as a loading control, which was used for normalization. (e) Double-label IF for GLAST (green) and CDC42EP4 (red) in WT cerebellar cortex showing their partial co-localization in Bergmann glial processes. Scale bars, 20 and 5 μm.
