Fig. 6
Measurement of the proximity of Slc7a5 and Kv1.2 with bioluminescence resonance energy transfer (BRET). a Emission spectra were collected from HEK293 cells transfected with Kv1.2-nanoluc + EGFP-Slc7a5 (green line normalized to peak). The Kv1.2-nanoluc (donor) spectrum was subtracted to yield the mEGFP component. Weighted components of the nanoluc (1.0) and mEGFP (0.1) spectra were used to fit the experimental spectrum (black dashed line). b mEGFP fluorescence (nanoluc-subtracted) was measured for Kv1.2-nanoluc co-expressed with various acceptor constructs, as indicated. c The area under the curve (AUC) for each BRET acceptor in b was normalized to the positive control AUC (Kv1.2-EGFP). Data are shown as mean ± SD (n = 4–5). ANOVA and a Tukey post hoc test were used to compare the BRET signals from EGFP-Slc7a5 (±Slc3a2) and EGFP-Slc1a5. d Western blot of dissociated cortical and hippocampal neurons from P2 rat pups at 14 days in vitro blotted with anti-Kv1.2 (left), anti-Slc7a5 (middle) and anti-β actin (right). e RT-PCR of Slc7a5 and Slc3a2 from dissociated cortical neurons from P1 rat pups after 4 days in vitro. f Cortical and hippocampal neurons from P2 rat pups at 7 days in vitro fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 and stained with anti-Kv1.2 and anti-Slc7a5 primary antibodies as in d and fluorescent secondary antibodies. Images are representative of three neuronal cultures. Scale bars represent 50 µM
