Anti ha

Cells were lysed on ice in 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 100 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, and 1% protease inhibitor cocktail (Sigma). Soluble extracts were incubated for 2 h at 4°C with relevant antibodies: anti-HA (Roche Applied Science) and a negative isotype control mouse immunoglobulin (IgG) (Santa Cruz Biotechnology), and complexes precipitated with protein A/G agarose (Santa Cruz Biotechnology). Western blotting was performed as described previously[22] (link), [40] (link)–[43] (link) using anti-HA (Roche), anti-V5 (Invitrogen), anti-PKCα, β, γ, ε, η, θ, ι, λ and δ (Cell Signaling Technology), anti-ERK1/2 (Cell Signaling Technology), anti-ERK1/2 phosphorylation (Cell Signaling Technology),anti-MEK(Cell Signaling Technology), and anti-GAPDH (Santa Cruz Biotechnology), and anti-Nox5 phosphorylation antibodies[21] (link).

Immunoblotting and immunoprecipitation were performed as previously described^{61 (link)}. The antibodies used in this study were: anti-dual phospho-JNK (Cell Signaling), anti-Flag (Sigma), anti-HA (Boehringer Mannheim), anti-phospho-Thr (Upstate), anti-phospho-Tyr (Upstate), anti-phospho-Ser (Upstate), anti-His (Sigma), anti-GST (GE), anti-JIP1 (Santa Cruz), and anti-myc (Upstate). In all cases, before performing gel-electrophoresis, the amount of total proteins was quantified using Bradford assay and equal amounts of proteins were loaded in each lane in a gel. All immunoblot analyses were independently performed at least three times.

After transfection for 24 hours, cells were lysed in a lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/mL leupeptin, 1% (v/v) glycerol and 0.1% (v/v) NP-40). Proteins in the samples were separated on a 10% SDS-PAGE and transferred to a nitrocellulose transfer membrane. The blots were blocked in 5% (w/v) skim milk for 1 hour and incubated with primary antibodies in TBST buffer overnight at 4°C. The antibodies used in this study were as follows: anti-phospho JNK (Cell Signaling), anti-Flag (Sigma), anti-HA (Boehringer Mannheim), and anti-myc (Upstate). Proteins were detected with the LAS-3000 (Fuji) imaging system using West Pico Chemiluminescent Substrate (Thermo Scientific) according to the manufacturer's instructions. Myc-tagged scaffold proteins were immunoprecipitated by incubation with an anti-myc antibody conjugated to agarose (Sigma) for 4 hours at 4°C. The samples were washed three times with lysis buffer and examined by western blot analysis. All the western blot analyses were performed at least three times.

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.

SDS-PAGE was performed using 8% and 12.5% acrylamide gels. For the Western blotting procedure, 0.2 μm nitrocellulose membranes were used. Following protein transfer to membranes, these were blocked in 5% fat-free milk powder, 5 mM NaN₃ and 0.1% Tween-20 in PBS.
Antibodies and their sources were: anti-HA (Roche, 15645900), anti-Cdc48 (own production), anti-α-tubulin (Abcam, YL1/2 MA1-80017), anti-Pma1 (Abcam, 40B7 Ab4645), anti-RGS-His (Qiagen, 34610), anti-Vinculin (Sigma, hVIN1 V9264), anti-GAPDH (Cell signalling technology, 14C10 2118), anti-Na/K ATPase α-1 (Merck, C464.6 05–369), anti-Hsp105 (HSPH1, Abcam, Ab108625), anti-HSPA4 (Abcam, Ab185219), anti-Ubiquitin (Dako, Z0458), anti-Myc (Chromotek, 9E1 9e1-100), anti-Rpn1 (Enzo Life Sciences, p112-1 PW9270), anti-20S α-subunits (Enzo Life Sciences, MCP231 PW8195), anti-Hsp70 (Invitrogen, 5A5 MA3-007), anti-Aspartoacylase (Thermo Scientific, PA5-29180), anti-GFP (Chromotek, 3H9 3h9-100). Secondary antibodies and their sources were: HRP-anti-rat (Invitrogen, 31470), HRP-anti-mouse (Dako, P0260), HRP-anti-rabbit (Dako, P0448).