Irdye 800cw streptavidin

The palmitoylation assay was performed according to Drisdel and Green (2004) (link). Imaginal tissues were dissected from 40 larvae or transfected S2 cells, homogenized, and suspended in 500 µl lysis buffer, pH 7.4, with 50 mM N-ethylmaleimide (Thermo Fisher Scientific). After rotating for 1–2 h at 4°C, lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Proteins were precipitated overnight by adding suitable antibodies to the supernatant at 4°C followed by addition of protein A or G beads for 4 h at 4°C. Afterward, the beads were treated with 1 M hydroxylamine (pH 7.0–7.2) or 1 M Tris, labeled using 1.0 µM BMCC-biotin (Thermo Fisher Scientific) in lysis buffer, pH 6.2, for ∼1–2 h at 4°C, and eluted with SDS sample buffer. For some of the Ft palmitoylation assays, immunoprecipitated proteins were treated with lambda phosphatase (25°C for 30 min). Protein samples were run on 8% or 10% SDS-PAGE and transferred to nitrocellulose membranes (LI-COR Biosciences), and biotinylation was detected with IR Dye 800CW Streptavidin (LI-COR Biosciences). Quantification of Ft palmitoylation was performed by calculating the ratio of the Streptavidin channel to total Ft protein in the immunoprecipitation lane, and then expressed relative to control (either Ft^ΔECD or Ft^{ΔECD-4623C/S}, depending on the experiment).