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. 2010 Apr 21;107(19):8830–8835. doi: 10.1073/pnas.0910644107

Fig. 2.

Fig. 2.

Ephrin-B3 knockdown reduces mEPSC frequency. (A) mEPSCs from whole-cell patch-clamp recordings of neurons expressing GFP plus indicated constructs (calibration, 40 pA, 1 s) and individual events (Inset: calibration, 20 pA, 20 ms). (B) Schematic of eB3 mutants used. HAeB3R is HA-tagged full-length eB3 rendered insensitive to eB3 shRNA#1. mCer-eB3/B1 is mCerulean tagged to the extracellular domain of eB3 fused to the intracellular domain of ephrin-B1. HAeB1/B3 is HA tagged to the extracellular domain of ephrin-B1 fused to a knockdown-insensitive intracellular domain of eB3. HAeB3R_L293A is HAeB3R in which alanine has replaced leucine 293 in a juxtamembrane domain (green star). (C) Quantification of mEPSC frequency after transfection with vector control (n = 14), ephrin-B1 shRNA (n = 11), eB3 shRNA#1, 1.5 μg/well (n = 23), eB3 shRNA#2 (n = 6), eB3 shRNA#1 plus HAeB3R (n = 19), eB3 shRNA#1 plus mCer-eB3/B1 (n = 12), eB3 shRNA#1 plus HAeB1/B3 (n = 6), or eB3 shRNA#1 plus HAeB3R_L293A (n = 7). *P < 0.005 from control. (D) Quantification of mEPSC amplitude after transfection with vector control (n = 686), ephrin-B1 shRNA (n = 566), ephrin-B1 shRNA plus HAeB1R (n = 861), eB3 shRNA#1 (n = 424), or eB3 shRNA#1 plus HAeB3R (n = 2979). ***P < 0.001. (E) Quantification of mEPSC frequency after transfection of 0.25 μg (n = 9) or 1.5 μg (n = 5) eB3 shRNA#1 per well. (F) Quantification of mEPSC frequency after transfection of eB3 shRNA#1 plus 0.25 μg (n = 8) or 1.0 μg (n = 13) HAeB3R per well. *P < 0.05. Error bars indicate SEM.