Irdye 680rd and 800cw secondary antibodies

Whole-cell lysates were prepared by scraping cells into ice cold RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 0.5% deoxycholate and 1% Triton X-100) containing protease and phosphatase inhibitors (Roche, Switzerland). Before loading onto 4–12% NuPAGE Bis-Tris gradient gels (Life Technologies), lysates were cleared by centrifugation. Following SDS-PAGE, proteins in gels were transferred to nitrocellulose using an iBlot (Life Technologies). Before blocking, proteins were fixed to the nitrocellulose by air drying the membrane overnight at room temperature. Blocking of membranes was then done in blocking buffer (Hepes buffered saline containing 0.1% Triton X-100, 1% BSA, 1% fish gelatin and 5 mM EDTA). Antibodies against Eps8 (BD Biosciences, Franklin Lakes, NJ; #610143), phospho-Erk (Cell Signaling Technology #4370),Erk (Cell Signaling Technology #9102), pMLC (Rockland, Limerick, PA; #600-401-416) and MLC (Rockland #600-401-938) were used at a 1:1000 dilution and incubated overnight at 4C in blocking buffer. IRDye 680RD and 800CW secondary antibodies (LI-COR Biosciences, Lincoln, NE) were then used at 1:5000 in blocking buffer for 2 hr at room temperature after extensive washing in Hepes Buffered Saline (HBS) containing 0.1% Triton X-100. Bands were then resolved on an Odyssey scanner (LI-COR Biosciences).

Fly heads were homogenized in Laemmli sample buffer (#1610737, Bio-Rad, Hercules, CA). For separation of polypeptides, samples were electrophoresed through precast polyacrylamide gels (Bio-Rad, #4561094) for 1 hour in a Tris/glycine/SDS buffer. Separated proteins were transferred to a nitrocellulose membrane and were then blocked with Odyssey blocking buffer (LI-COR Lincoln, NE, #927–40000) for 1 hour prior to incubation overnight with rabbit anti-pAMPKα (1:1000, Cell Signaling technology, Danvers, MA, #2535) and mouse anti-α-Tubulin (Sigma #T9026, diluted 1:5000) antibody at 4 °C. After 3 x 15-minute washes in PBS at RT, samples were incubated with IRDye 680RD and 800CW secondary antibodies diluted 1:10,000 (LI-COR) for 30 minutes at RT. Western blots were imaged using an Odyssey Fc imaging system (LI-COR) and ImageJ was used to quantify probe signal intensity.