Knockdown of α2δ1 and α2δ3 Subunits

For knockdown of the α2δ1 subunit, siRNA target sequences corresponding to the α2δ1 coding region (CACNA2D1, GenBank accession number NM_009784.2) (Obermair et al., 2005 (link)) were selected and tested for efficient knockdown. The siRNA was expressed as shRNA under the control of a U6 promoter (derived from the pSilencer1.0-U6 siRNA expression vector, Ambion) cloned into the pβA-eGFP plasmid (Obermair et al., 2010 (link)). For lentiviral expression, α2δ1 shRNA was cloned into pHR as previously described (Subramanyam et al., 2009 (link)). For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428). Based on their specificity for rat and mouse α2δ3, two of these constructs were tested for their knockdown efficiency where the construct “C” was evaluated to results in a reduction of α2δ3 expression down to 40%-50%. As control for α2δ-knockdown experiments, a noneffective 29-mer scrambled shRNA cassette cloned into pGFP-C-shLenti Vector (catalog #TR30021; OriGene Technologies) was used.