Sic001 1nmol

Isoform-specific siRNA oligonucleotides that target the F-actin–binding domain in rat AMOT-130 were obtained from Ambion/Thermo Fisher Scientific and Sigma-Aldrich. The target sequences were: si130#1, 5′-GTCCGATCCTTGAGCGAAA-3′ (s152896; Ambion), and si130#2, 5′-CCAAGATGAAGGCCTTAGA-3′ (SASI_Rn02_00284990; Sigma-Aldrich). A CsiRNA whose sequence was not homologous to any vertebrate sequence was used as negative control (SIC001-1NMOL; Sigma-Aldrich). To evaluate the knockdown efficacy of endogenous AMOT-130, 20 µM siRNA were transfected to a final concentration of 10 nM in rat IEC-18 cells (passage 5–10) using Lipofectamine 2000 (LF2000; Invitrogen). Cells were harvested 4 d later, and the relative expression levels of endogenous AMOT-130 were analyzed by immunoblotting using anti–AMOT-130 antibody. Quantification of the Western blot membranes by densitometry estimated a reduction in the AMOT-130 levels to ∼90% in IEC-18 cells relative to controls (see Fig. 3 A). For knockdown experiments, neurons at 11 DIV were transfected with siRNA together with 5 µg pEGFP–β-actin plasmid using LF2000 and prepared for experiments at 15–16 DIV. Alternatively, 2.5 × 10⁶ dissociated hippocampal neurons were electroporated with 1 µM si130#1 or CsiRNA at the day of plating (0 DIV) and analyzed by immunoblotting at 9 DIV.