Journal: Nature Communications

Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

doi: 10.1038/ncomms10090

Figure Lengend Snippet: ( a ) Quantitative co-IP/IB assay between SEPT4 and SEPT7, SEPT2 or GLAST in WT and KO cerebellar lysates. While the interaction among the septin subunits did not differ, the relative amount of GLAST pulled down with SEPT4 from KO lysate was significantly less than that from WT ( n =3, * P <0.05, NS, P >0.05 by t -test), indicating that septin–GLAST interaction depends on CDC42EP4. See for the comparable amount and solubility of these proteins in WT and KO cerebella. ( b ) Double-label IF for GLAST (green) and a Purkinje cell marker Car8 (red) in WT and KO cerebellar cortices. Genetic loss of CDC42EP4 caused no recognizable difference in the distribution of GLAST up to the resolution. Scale bars, 20 and 1 μm. ( c ) Immunoelectron microscopy images for GLAST in WT and KO molecular layers. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. The pattern of GLAST distribution appears comparable to that of a previous study . Scale bar, 200 nm. ( d ) Quantitative analysis of immunoelectron microscopy data. Bergmann glia selectivity and labelling density of GLAST were comparable between WT and KO mice: Bergmann glia; 22.4±1.3/21.1±1.2 particles per μm ( n =519/467 particles from two littermate pairs, NS, P >0.05 by Mann–Whitney U -test). Postsynaptic membrane; 0.50±0.21/0.68±0.24 particles per μm ( n =5/8 particles, P =0.68 by Mann–Whitney U -test; cf . and ). ( e ) Cumulative histogram of the distance of GLAST measured from the nearest PSD edge (for example, arrowheads in c ). A significant right shift of the curve for KO mice demonstrates delocalization of GLAST away from PSDs of PF–PC synapses (median, WT=0.27 μm, KO=0.31 μm from the nearest edge of PSD; n =22/19 synapses from two littermates for each genotype, *** P <0.001 by Kolmogorov–Smirnov test).

Article Snippet: We used antibodies for septins, SEPT2 (1:2,000), SEPT4 (1:3,000) and SEPT7 (1:4,000) as previously described , GLAST , 3-phosphoglycerate dehydrogenase (Phgdh) , calbindin , carbonic anhydrase 8 (Car8) , VGluT1, 2 (ref. ) and commercial antibodies for GluR1, 2 (Alomone Labs, AGC-004, 1:200, AGC-005, 1:150), GLAST (Frontier Institute, Rb-Af660, 1:2,000), PSD-95 (Cell Signaling, 3450, 1:1,000), CDC42 (Santa Cruz, L0809, 1:100), β-actin (Sigma, A5441, 1:5,000) and α-Tubulin (Sigma, T9026, 1:10,000).

Techniques: Co-Immunoprecipitation Assay, Solubility, Marker, Immuno-Electron Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

doi: 10.1038/ncomms10090

Figure Lengend Snippet: Proteomic analysis for the binding partners of CDC42EP4 in the cerebellum.

Article Snippet: We used antibodies for septins, SEPT2 (1:2,000), SEPT4 (1:3,000) and SEPT7 (1:4,000) as previously described , GLAST , 3-phosphoglycerate dehydrogenase (Phgdh) , calbindin , carbonic anhydrase 8 (Car8) , VGluT1, 2 (ref. ) and commercial antibodies for GluR1, 2 (Alomone Labs, AGC-004, 1:200, AGC-005, 1:150), GLAST (Frontier Institute, Rb-Af660, 1:2,000), PSD-95 (Cell Signaling, 3450, 1:1,000), CDC42 (Santa Cruz, L0809, 1:100), β-actin (Sigma, A5441, 1:5,000) and α-Tubulin (Sigma, T9026, 1:10,000).

Techniques: Binding Assay, Sequencing

Journal: Nature Communications

Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

doi: 10.1038/ncomms10090

Figure Lengend Snippet: ( 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 . 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.

Article Snippet: We used antibodies for septins, SEPT2 (1:2,000), SEPT4 (1:3,000) and SEPT7 (1:4,000) as previously described , GLAST , 3-phosphoglycerate dehydrogenase (Phgdh) , calbindin , carbonic anhydrase 8 (Car8) , VGluT1, 2 (ref. ) and commercial antibodies for GluR1, 2 (Alomone Labs, AGC-004, 1:200, AGC-005, 1:150), GLAST (Frontier Institute, Rb-Af660, 1:2,000), PSD-95 (Cell Signaling, 3450, 1:1,000), CDC42 (Santa Cruz, L0809, 1:100), β-actin (Sigma, A5441, 1:5,000) and α-Tubulin (Sigma, T9026, 1:10,000).

Techniques: Co-Immunoprecipitation Assay, Lysis