Primary cerebellar granule neurons were treated with lentivirus carrying TMEM163 shRNA or a random sequence in parental pFUGW (Ctrl) as well as C-terminally GFP-tagged TMEM163 mutant resistant to the shRNA (TMEM163mutGFP).
(A) Total protein amounts of TMEM163 and other proteins were measured in primary cerebellar granule neurons treated with shRNA lentivirus. For comparison, we loaded 50% of the control (Ctrl) lysate.
(B) Quantitation of total protein amounts. TMEM163 shRNA significantly reduced TMEM163 protein compared with control shRNA. Total protein amounts of GluN1 and α-tubulin were unaltered (n = 4).
(C and D) Agonist-evoked whole-cell currents were measured under whole-cell configuration (Vh = −70 mV) with the cell capacitance between 4.0–4.3 pF. Representative traces and quantitation of peak amplitudes of 100 μM ATP-evoked currents (C) or 100 μM Glu-evoked currents (D) upon 6-s application on neurons with a GFP signal from GFP or TMEM163mutGFP (n = 15–22). Artifacts of a 150 ms/2 mV pulse for cell capacitance measurement were omitted from traces shown.
Data are mean ± SEM. Student’s t test (B) and one-way ANOVA, followed by Bonferroni’s post-test (C); *p < 0.05; ***p < 0.001.