Supplementary Figure 3: Specificity of the phospho-Ser332 antibody.
From: Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3
(a) Alignment of cytoplasmic regions of all three ephrin-B family members, demonstrating a high degree of homology. Phosphorylable S332 residue found within a putative MAPK phosphorylation sequence PPQSPP is shown in the red box. Highlighted in green is the peptide that was used to generate and affinity purify the phospho-S332 (pS332) antibody.
The phospho-S332 (pS332) antibody appears to be selective because: 1) the antibody recognized ephrin-B3 in HEK 293T cells, but not ephrin-B1, ephrin-B2 or a non-phosphorylable ephrin-B3 (S332A) mutant (panel S3b); 2) the 37 kDa band corresponding to phospho-S332 is absent in brains from ephrin-B3 null mice (panel S3c); 3) treatment of neuronal lysates with calf intestinal phosphatase (CIP) resulted in the loss of the phospho-S332 band (panel S3d); and 4) immunostaining of cultured neurons following knockdown of ephrin-B3 (panels S3e-f) or of brain sections from Efnb3–/– mice (panels S3g-h) show significantly reduced levels of staining.
(b) Western blots showing that the phospho-S332 (pS332) antibody specifically recognizes WT ephrin-B3. Lysates from HEK 293T cells transfected with ephrin-B1 (HA-eB1), ephrin-B2 (HA-eB2), ephrin-B3 (Flag-eB3), or an ephrin-B3 with a mutation to S332 rendering it non-phosphorylatable (Flag-eB3_S332A). Only wild type ephrin-B3 is recognized by the pS332 antibody (arrow). Bottom blots were probed with α-HA and α-Flag to validate the expression of transfected ephrin-B constructs in HEK 293T cells.
(c) Western blots from WT and ephrin-B3 null (Efnb3–/–) cortical lysates probed with pS332 antibody. Arrow indicates the band of appropriate molecule weight is present only in WT lysates. The pS332 signal is absent from Efnb3–/– cortical lysates (arrow).
(d) Western blots of DIV14 cortical neuron lysates probed with the pS332 antibody treated with calf intestinal phosphatase (CIP, 1unit/µg of protein) for one hour. CIP treatment eliminates pS332 specific signal (arrow). Total ephrin-B3 expression (bottom blot, arrow) is not changed by CIP treatment.
(e) Representative images of dendritic segments from cortical neurons co-transfected with GFP and either control or ephrin-B3 shRNA constructs at DIV0. Neurons were stained with the pS332 antibody at DIV21. Arrows indicate pS332 puncta along GFP positive dendritic shaft. Sale bar: 3 µm.
(f) Knockdown of endogenous ephrin-B3 with shRNA causes a significant reduction in pS332 puncta density. Quantification of average density of pS332 ephrin-B3 puncta along dendrites of cultured neurons. Data are represented as the mean ± s.e.m., control (n = 5), ephrin-B3 shRNA (n = 6) neurons. ***p<0.001, un-paired t-test (two-tailed), t=9.181, df=9.
(g) P14 cortical sections from wild type and ephrin-B3 null (Efnb3–/–) mice stained with pS332 antibody. Prominent labeling is seen in wild type cortex, but this signal is absent in sections from ephrin-B3 null (Efnb3–/–) cortex. Scale bar: 200 µm.
(h) Representative images of apical dendrites of GFP labeled cortical neurons from wild type and ephrin-B3 null (Efnb3–/–) sections. Prominent pS332 labeling is observed in the shaft of wild type neurons, but this signal is absent in neurons from ephrin-B3 null brain. Scale bar: 3 µm.
