Figure 1. Knockdown of endogenous CASK prevents dendritic spine formation in cultured hippocampal neurons. (A) CASK shRNA reduced the expression of endogenous CASK in HEK cells. The CASK shRNA construct was transfected into HEK cells. 1 d later, cells were harvested for immunoblotting analysis using CASK and tubulin antibodies. Nonsilencing construct was used as a negative control. Tubulin was used as an internal control. (B) CASK shRNA down-regulated overexpressed Myc-tagged rat CASK but not Myc-tagged PSD-95, Myc-tagged SAP-97, or a Myc-tagged rat CASK silent mutant insensitive to CASK shRNA. COS cells were cotransfected with CASK shRNA or nonsilencing construct and various MAGUK protein expression plasmids as indicated and were harvested 1 d later for immunoblotting using Myc tag antibody. Tubulin was used as an internal control. (C) Immunostaining of cultured hippocampal neurons showing the reduction of endogenous CASK by CASK shRNA. Cultured hippocampal neurons were cotransfected with CASK shRNA construct and GFP-actin at 14 DIV. 2 d later, cells were stained with CASK antibody. Two representative images are shown. Arrowheads point to the transfected neurons, which are GFP-actin positive. Higher magnifications of the boxed areas in the middle panel are shown on the right. More images showing the knockdown effect of CASK shRNA are included in Fig. S1 (available). (D) CASK shRNA prevented dendritic spine formation. Cultured hippocampal neurons were cotransfected with GFP-actin and CASK shRNA, nonsilencing control, or CASK mutant resistant to CASK shRNA as indicated at 13–14 DIV. 5 d later, cells were harvested for fluorescence immunostaining using CASK (visualized by AlexaFluor594) and GFP (visualized by AlexaFluor488) antibodies. Only the GFP patterns are shown. (E) Quantification of the effect of CASK shRNA on dendritic spine morphology. For each experiment, at least 11 transfected neurons were analyzed from two independent experiments. A total of 1,027 (nonsilencing), 798 (CASK shRNA), and 549 (CASK shRNA + CASK mutant) spines were analyzed. Mean values ± SEM for each group are shown. Bars: (C) 20 μm; (D) 1 μm.
Figure 1.

Knockdown of endogenous CASK prevents dendritic spine formation in cultured hippocampal neurons. (A) CASK shRNA reduced the expression of endogenous CASK in HEK cells. The CASK shRNA construct was transfected into HEK cells. 1 d later, cells were harvested for immunoblotting analysis using CASK and tubulin antibodies. Nonsilencing construct was used as a negative control. Tubulin was used as an internal control. (B) CASK shRNA down-regulated overexpressed Myc-tagged rat CASK but not Myc-tagged PSD-95, Myc-tagged SAP-97, or a Myc-tagged rat CASK silent mutant insensitive to CASK shRNA. COS cells were cotransfected with CASK shRNA or nonsilencing construct and various MAGUK protein expression plasmids as indicated and were harvested 1 d later for immunoblotting using Myc tag antibody. Tubulin was used as an internal control. (C) Immunostaining of cultured hippocampal neurons showing the reduction of endogenous CASK by CASK shRNA. Cultured hippocampal neurons were cotransfected with CASK shRNA construct and GFP-actin at 14 DIV. 2 d later, cells were stained with CASK antibody. Two representative images are shown. Arrowheads point to the transfected neurons, which are GFP-actin positive. Higher magnifications of the boxed areas in the middle panel are shown on the right. More images showing the knockdown effect of CASK shRNA are included in Fig. S1 . (D) CASK shRNA prevented dendritic spine formation. Cultured hippocampal neurons were cotransfected with GFP-actin and CASK shRNA, nonsilencing control, or CASK mutant resistant to CASK shRNA as indicated at 13–14 DIV. 5 d later, cells were harvested for fluorescence immunostaining using CASK (visualized by AlexaFluor594) and GFP (visualized by AlexaFluor488) antibodies. Only the GFP patterns are shown. (E) Quantification of the effect of CASK shRNA on dendritic spine morphology. For each experiment, at least 11 transfected neurons were analyzed from two independent experiments. A total of 1,027 (nonsilencing), 798 (CASK shRNA), and 549 (CASK shRNA + CASK mutant) spines were analyzed. Mean values ± SEM for each group are shown. Bars: (C) 20 μm; (D) 1 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal