Figure 3.
JAK2 Knockdown Blocks the Induction of NMDAR-LTD
(A) The efficiency of two JAK2 shRNAs, compared to one control shRNA, was assessed in HEK cells cotransfected with a plasmid coding for JAK2. The values shown are relative to the ratio JAK2/GAPDH obtained from the cells transfected with JAK2 only. ∗significantly reduced compared to untransfected cells or the control shRNA.
(B) Images of cultured neurons transfected with a plasmid coding for GFP and the control or JAK2 shRNA, as indicated, and labeled with a JAK2 antibody (red). Scale bars: 10 μm.
(C–E) AMPAR-mediated EPSCs (EPSCA) and NMDAR-mediated EPSCs (EPSCN) were recorded in cells transfected (Trans) with a JAK2 shRNA or the control shRNA, as indicated, and nearby untransfected cells (Untrans). Peak amplitudes (EPSCA, at −70 mV) or the amplitude measured 60 ms poststimulation (ESPCN, at +40 mV) were plotted for each pair (black circles). Gray symbols represent mean ± SEM. Insets show representative traces, which are averages of three successive records. Calibration bars for all traces shown: 40 pA/20 ms.
(F–H) The NMDAR-LTD induction protocol failed to induce LTD of EPSCA in cells transfected with the shRNAs for JAK2 (n = 7 and 6 for the first and second JAK2 shRNA tested; F and G) while an input-specific LTD was consistently observed in cells transfected with the control shRNA (n = 8; H). The NMDAR-LTD induction protocol is marked by a horizontal line. Insets show representative traces of the test (black circle) and control (white circle) inputs, before and 25–30 min after LTD induction; each trace is an average of three successive EPSCs. Calibration bars: 50 pA/40 ms.