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. 2016 Apr 21;11(4):e0153009. doi: 10.1371/journal.pone.0153009

Fig 6. Fluorescent labeling of pacSph metabolites to study protein lipid interaction.

Fig 6

Flag-tagged STARD7 (A) and p24 (B) were expressed in HeLa and HeLa ΔSGPL1 cell lines. Cells were metabolic labeled with 5 μM pacSph (STARD7) or 0.5 μM pacSph with or without sphingosine (p24) for 7 h and then UV irradiated to cross-link pacSph metabolites to nearby protein. Protein lysates were subjected to click reaction with Alexa647 azide (shown in red) and the ectopically expressed proteins were immunoprecipitated. After SDS-PAGE and immunoblot with fluorescently labeled secondary antibodies (shown in green), lipid and protein signals were detected in separate channels. Lys, Lysate, IN, input of immunoprecipitation, IP, immunoprecipitated material.