Figure 7. Silencing of neuronal activity by potassium withdrawal leads to LZK-dependent activation of JNKs in cerebellar granule neurons.

(A) CGNs from wild-type mice were maintained in media containing 25 mM KCl for five days. Potassium withdrawal was performed by switching to media containing 5 mM KCl without serum as previously described³⁹ on day 6. CGNs were collected at the indicated times following potassium withdrawal and total cell lysates were immunoblotted for the indicated endogenous proteins. (B) LZK^T/T CGNs were infected with AAV-GFP or AAV-GFP-Cre 40 hours after plating. Top panel, total cell lysates collected on 0, 3, 6 DIV were immunoblotted for endogenous LZK to assay LZK depletion in the total cell population. Immunoblot-based quantification of signal intensity (S.I.) of endogenous LZK protein levels normalized to that of β-actin is shown. Bottom panel, AAV-GFP or AAV-GFP-Cre infected LZK^T/T CGNs were subjected to potassium withdrawal on 6 DIV. Total cell lysates collected at the indicated times following treatment were immunoblotted for the indicated endogenous proteins. Two different exposures (low and high) for p-JNK2 and p-JNK1 are shown for better comparison. *JNK 54 kDa isoform; **JNK 46 kDa isoform. (C) Graphs quantify endogenous p-JNK protein levels from the bottom panel in (B). Immunoblot-based quantification of JNK protein expression was first normalized to β-actin in the corresponding sample, and shown as a value relative to the control (presented as baseline of 1). (D) LZK^T/T CGNs were infected with control AAV-GFP or AAV-GFP-Cre 40 h after plating. AAV-infected LZK^T/T CGNs were subjected to potassium withdrawal on 6 DIV. Total cell lysates collected at the indicated times following treatment were immunoblotted for the indicated endogenous proteins.