Figure 6. Silencing endogenous AKAP150 reduces AMPARs-GluA1 subunit levels. A, AKAP150 was silenced (sh or shAKAP150) or overexpressed (ov or ovAKAP150) in neuronal cultures after transduction with lentiviral vectors. Representative blot images showing the efficiency of shAKAP150 or ovAKAP150 transduction in primary neurons. Empty lentiviral vector (Ø; pLVTHM or pWPI) was used as a control. B, Silencing AKAP150 does not affect related scaffold proteins like PSD95 (∼95-kDa band) or SAP97 (∼140-kDa band). Representative blot images showing levels of AKAP150 (top panel; as a control of silencing or overexpressing), PSD95 and SAP97 when endogenous AKAP150 was knocked down (shAKAP150) or overexpressed (ovAKAP150). GAPDH (bottom panel) was used as a loading control. C, Quantification of PSD95 or SAP97 levels when AKAP150 was silenced (shAKAP150) or overexpressed (overAKAP150) normalized to basal (n = 4). D, Endogenous AKAP150 silencing reduces S845-GluA1 phosphorylation and total GluA1 protein. Representative blot images showing AKAP150 levels (top panel; as a control of silencing), phosphorylated GluA1 (pS845-GluA1), total GluA1 and GAPDH (bottom panel; loading control). E, Quantification in pS845-GluA1 and GluA1 levels in response to AKAP150 knock-down compared with basal (n = 3, *p = 0.0057 vs basal; #p = 0.0070 vs basal). F, AKAP150 overexpression does not modify S845-GluA1 phosphorylation and total GluA1 protein levels. Representative blot images showing AKAP150 (top panel; as a control of overexpression), phosphorylated GluA1 (pS845-GluA1), total GluA1 and GAPDH (bottom panel; loading control) levels. G, Quantification of pS845-GluA1 and GluA1 in response to AKAP150 overexpression compared with basal (n > 3). H, Representative confocal images of dendrites show GluA1 (magenta), presynaptic VGlut1 (gray), and postsynaptic PSD95 (blue) levels after knocking down AKAP150 compared with basal condition (Ø; empty vector). GFP expression (green) is used as a control of transduction. Down-regulation of endogenous AKAP150 reduced dendritic and synaptic GluA1 intensity. Bottom panels in H show the merge for GFP/GluA1/PSD95/VGlut1. I, Quantification of GluA1 (GluA1) intensity normalized to basal. AKAP150 knockdown expression reduced dendritic (o) and synaptic (ν) GluA1 (n > 30 dendrites of different neurons from five independent cultures, *p < 0.0001 vs basal; #p < 0.0001 vs basal). J, Surface GluA1 levels on cLTD in presence of shAKAP150. Representative blot images showing decreased levels of surface GluA1 after cLTD in presence of shAKAP150 (sGluA1; ∼110-kDa band, top panel) related to total GluA1 (tGluA1; ∼110-kDa band, middle panel). GAPDH was used as a loading control (∼36-kDa band, bottom panel). K, Quantification of surface GluA1 changes in response to cLTD in presence of shAKAP150 compared with basal condition (n = 3, *p = 0.0030 for cLTD, *p = 0.0012 for shAKAP150+cLTD, *p = 0.0018 for empty+cLTD and *p = 0.0141 for shAKAP150 vs basal). L, Quantification of AKAP150 in response to cLTD in presence of shAKAP150 compared with basal condition (n = 3, *p = 0.0080 for cLTD, *p < 0.0001 for shAKAP150+cLTD, *p = 0.0080 for empty+cLTD and *p = 0.0003 for shAKAP150 vs basal). Scale bars = 5 μm. Bars represent mean ± SEM.