α2δ 1 ab

Ca channel Abs used were: α₂δ-1 Ab (mouse monoclonal against α₂-1 moiety, Sigma-Aldrich, epitope identified in (Cassidy et al., 2014 (link))), anti-Ca_V2.2 II-III loop Ab (rabbit polyclonal) (Raghib et al., 2001 (link)). A bespoke, affinity-purified Cachd1 rabbit polyclonal Ab was raised by Cambridge Research Biochemicals (Billingham, UK) against the predicted extracellular domain of zCachd1 protein, produced by transient transfection of mammalian cells (Durocher et al., 2002 (link)) (G.T.P., S.W.W., and Gavin J. Wright, unpublished data). Purified Ab activity was confirmed by enzyme-linked immunosorbent assay. Other Abs used were anti-HA (rat monoclonal, Roche), anti-HA (rabbit polyclonal, Sigma), anti-GAPDH Ab (mouse monoclonal, Ambion), and GFP Ab (Living Colors, rabbit polyclonal; BD Biosciences). For immunocytochemistry, secondary Abs (1:500) used were anti-rabbit-Alexa Fluor 594, anti-rat-Alexa Fluor 488, anti-mouse-Alexa Fluor 647 (Life Technologies) or anti-rat fluorescein isothiocyanate (Sigma-Aldrich). The secondary Abs used for Western Blotting were goat anti-rabbit, goat anti-rat, and goat-anti-mouse Abs coupled to horseradish peroxidase (HRP) (Biorad). ω-conotoxin GVIA was purchased from Alomone, and applied by local perfusion.

Ca channel antibodies (Abs) used were: α₂δ-1 Ab (mouse monoclonal against α₂-1 moeity, Sigma-Aldrich, epitope identified in [Cassidy et al., 2014 (link)]), α₂-3 (71–90) Ab (rabbit; polyclonal) (Davies et al., 2010 (link)), δ-3 (1035–1049) Ab (Davies et al., 2010 (link)), anti-Ca_V2.2 II-III loop Ab (rabbit polyclonal) (Raghib et al., 2001 (link)). Other Abs used were anti-HA (rat monoclonal, Roche), anti-HA (rabbit polyclonal, Sigma), anti-GAPDH Ab (mouse monoclonal, Ambion), anti-flotillin-1 (mouse monoclonal, BD Biosciences), and GFP Ab (Living Colors, rabbit polyclonal; BD Biosciences). For immunocytochemistry, secondary Abs (1:500) used were anti-rabbit-Alexa Fluor 594, anti-rat-Alexa Fluor 594, anti-mouse-Alexa Fluor 647 (Life Technologies) or fluorescein isothiocyanate (FITC)-anti-rat (Sigma-Aldrich). The following secondary Abs were used for western blot: goat anti-rabbit, goat anti-rat and goat anti-mouse Abs coupled to horseradish peroxidase (HRP) (Biorad, Hemel Hempstead, UK). The signal was obtained by HRP reaction with fluorescent product (ECL 2; Thermo Scientific) and membranes were scanned on a Typhoon 9410 phosphorimager (GE Healthcare). Lyophilized active thrombin was obtained from Sigma, suspended in filter-sterilised PBS (Sigma; pH7.4) to 1000 U/ml and frozen in aliquots until use.