Sds express plus page gel

Protein samples had SDS urea lysis buffer and 2-mercaptoethanol added, and were immediately boiled for 5 min. After boiling, samples were vortexed for 2 min before being transferred to ice. Samples were run on a 10% SDS Express plus PAGE gel (#M01012; GenScript) using SDS-MOPS buffer. Proteins were transferred to a nitrocellulose membrane in tris–gylcine buffer, washed briefly in 1× PBS–tween, and blocked with 5% dehydrated milk in 1× PBS for 1–2 h. 1× PBST was used as wash buffer. Primary antibodies were diluted in 1× PBS–tween + milk overnight at 4°C. The following morning, the membrane was washed three times using 1× PBS–tween before 2 h incubation with secondary antibody, followed by three more washes. Using WesternBright ECL (#K-12045-D20; Advansta), blots were exposed using the LI-COR Odyssey Infrared Imaging System. Antibodies used were as follows:

Whole worm lysis was flash frozen in SDS urea lysis buffer and 2-mercaptoethanol (S3A, S3B & S3E Fig), or whole worm lysis was frozen and incubated with SDS urea lysis buffer and 2-mercaptoethanol during thaw (S3C, S3D & S3F Fig). Samples were boiled and run on a 10% SDS Express plus PAGE gel (#M01012; GenScript). 1X PBS-Tween was used as wash buffer. Antibodies used were as follows: α-V5 (1:2,000- S3A, S3B & S3E Fig, #46–0705; 1:5,000- S3C, S3E & S3F Fig, R960-25; Invitrogen), α-FLAG (1:3,000; #F1804, Sigma-Aldrich), and α-tubulin (1:1,000- S3A, S3B & S3E Fig; 1:10,000- S3C, S3D & S3F Fig; ab1157911, Developmental Studies Hybridoma Bank) used as loading control. Secondary antibodies used were α-mouse antibody conjugated to HRP (1:10,000- Sup 3A, B, & E; 1:5,000- Sup 3C, D, & F). 5% milk in 1X PBS-Tween was used for blocking (S3A, S3B & S3E Fig; no blocking was used in S3C, S3D & S3F Fig). WesternBright ECL (#K-12045-D20; Advansta) was used for HRP detection and blot development. The LI-COR Odyssey Infrared Imaging System was used for development and analysis of western blots. For proteasome inhibition 100 adult worms were incubated in 1μM MG132 (EMD Millipore) for 8 hours, 20-24hrs after L4 stage selection. These worm incubations were then used for western blot analysis.