Fig. 1.

A chemical screen for mitochondrial ceramide-binding proteins yields VDAC1 and -2. a Structure of the photoactive and clickable C₁₅-ceramide analog, pacCer. b Mitochondria isolated from HeLa cells were incubated with liposomes containing increasing amounts of pacCer, UV irradiated, and then click reacted with AF647-N₃. Samples were processed for SDS-PAGE, subjected to in-gel fluorescence (IGF, red), and stained with Coomassie blue (CB, blue). p33•Cer denotes a prominently photolabeled protein band of ~33 kDa. c Strategy for the identification of p33•Cer. d p33•Cer was purified from pacCer-labeled and TAMRA/biotin click-reacted mitochondria using NeutrAvidin-beads, imaged by IGF, excised from the gel, digested by trypsin, and then identified by LC-MS/MS. Data from two independent experiments revealed that p33•Cer corresponds to VDAC1 and VDAC2. e Specificity of anti-VDAC antibodies was validated by immunoblotting of mitochondria isolated from HeLa cells treated with non-silencing (siNS) or VDAC-targeting siRNAs (siVDAC1, siVDAC2). Note that the anti-VDAC3 antibody cross-reacts with VDAC2. f Mitochondria isolated from siVDAC1/2-treated HeLa cells were photolabeled with pacCer, click-reacted with AF647-N₃, and subjected to IGF analysis followed by CB staining. g Fractions obtained during affinity purification of p33•Cer were subjected to SDS-PAGE, transferred on nitrocellulose, analyzed by on-blot-fluorescence (OBF) and probed with antibodies against VDAC1, TOM40, and p60-Mito. h Fractions obtained during affinity purification of p33•Cer were processed as in g and probed with anti-VDAC2 and anti-VDAC3 antibodies. T total mitochondria extract, FT flow-through, W wash, E eluate