Western Blot Analysis of Protein Complexes

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).