Measuring Protein Synthesis and Signaling

Whole-cell extracts from cell lines and islet lysates were prepared and subjected to immunoblot analysis as described previously^{11 (link)}. For immunoblotting of puromycin incorporation into protein, puromycin was added to the culture medium at 1 ug/mL during the final 15 minutes of incubation. Cells were then washed twice with cold PBS and lysed. Immunoblot analyses were performed after separation of protein extracts on a 4% to 20% gradient SDS-polyacrylamide gel and were visualized using fluorescently labeled primary antibody to puromycin (mouse anti-puromycin, 1:1000 concentration, Kerafast, Catalog number EQ0001), p-4EBP1 (rabbit anti-p-4EBP1, 1:1000, Cell Signaling Technology, number 9455), p-p70 S6K (Thr389) (rabbit anti-p-p70S6K, 1:1000, Cell Signaling Technology, number 2708), p-eIF2α (rabbit anti-p-eIF2 α, 1:1000, Cell Signaling Technology, number 9721), and secondary antibodies (Li-Cor Biosciences) and quantified using a Li-Cor Odyssey scanner and Image J 1.38x^{50 (link)}.