Fig. 7.

Protein 4.1B enhances NMDAR localization at synapses. (A) Transfected hippocampal neurons were incubated with antibodies to GFP (rb) at 4°C to label surface GFP-NR2B (surface, red) and subsequently fixed, permeabilized and reincubated with GFP antibodies (ms) to reveal total GFP-NR2B (total, green) and antibodies to synapsin I (not shown). Scale bar = 50 μm. (B) Quantification of the intensity of surface GFP-NR2B puncta at synaptic and non-synaptic sites in hippocampal neurons expressing shRNA to 4.1B (4.1B shRNA), control shRNA (Ctl shRNA), 4.1B and 4.1B ΔFERM and empty vector (pCDNA3) for 24 hours. Error bars represent s.e.m. (* p < 0.05). (C) Neurons expressing shRNA to 4.1B (4.1B shRNA) and GFP, control shRNA (Ctl shRNA) and GFP, 4.1B-GFP, 4.1B ΔFERM-GFP or GFP alone for 48 hours were immunolabeled with antibodies to synapsin I and PSD-95. Scale bar = 50 μm. (D) Quantification of the numbers of synapses per 20 μm of dendrite as determined by the colocalization of synapsin I and PSD-95. Synapse number was normalized to control conditions. Error bars represent s.e.m. (* p < 0.05, n = 11).