Anti rabbit 700

Two-hundred young adult worms were grown at 20° on 100 mm OP50-seeded plates for one generation and collected by washing in M9 buffer. Each worm pellet was resuspended in 1× SDS loading buffer (2 μl/mg of pellet) and heated in a microwave (4 × 20 sec with 1 min cooling). Lysates were then centrifuged (15,000 × RCF, 10 min) and supernatant was transferred into new tubes. Next, 10 µl of worm lysate was loaded per well and analyzed by standard western blot. SEP-1 was detected by using a polyclonal rabbit antibody (Richie et al. 2011 (link)) at a dilution of 1:750. Actin was detected using the mouse monoclonal antibody C4 from Millipore (Temecula, CA) at a dilution of 1:5000. Secondary antibodies used were anti-rabbit 700 and anti-mouse 700 from Li-Cor. Quantification was done using Image Studio software. Actin was used to normalize signals between lanes. All antibody incubations were done in the presence of 5% (w/v) nonfat milk.

Quadriceps muscle was homogenized as whole tissue (~30 mg) and mitochondrial isolations (~80 mg) for immunoblotting as previously described (Newsom et al. 2017). Approximately 35 μg of protein from tissue or cell lysates was resolved on 7–12% Bis‐Tris gels and transferred to nitrocellulose membranes. Two control samples (20 μg of insulin‐stimulated L6 cell samples) were loaded onto each gel and their average intensity was used to normalize intensities between gels. Ponceau staining of membranes was used to verify equal loading and transfer of protein to the nitrocellulose membrane. Images were generated using infrared detection (Odyssey, Licor, Lincoln NE). Primary antibodies included Akt (no. 2920; Cell Signaling Technology), pAkt^Ser473 (no. 9271; Cell Signaling Technology), LC3 (no. 12741; Cell Signaling Technology), p62 (no. 7695; Cell Signaling Technology), mTOR (no. 4517; Cell Signaling Technology), pmTOR^Ser2448 (no. 2971; Cell Signaling Technology), pUlk1^Ser757 (no. 6888; Cell Signaling Technology), AMPK (no. 5832; Cell Signaling Technology), pAMPK^Thr172(no. 2535; Cell Signaling Technology). Secondary antibodies included anti‐rabbit‐700 (1:10,000; no. 926‐68071; Licor) and anti‐mouse‐800 (1:50,000; no. 926‐32212; Licor).