Protein extraction for Western blotting

Worms (±18,000 L4 in liquid culture) were pelleted, washed twice in M9 (42.2 mM Na₂HPO₄, 22 mM KH₂PO₄, 85.7 mM NaCl, and 1 mM MgSO₄), and frozen. Thawed worms were resuspended in 4× Laemmli Sample Buffer [200 mM tris (pH 8.5), 8% SDS, and 40% glycerol], boiled for 5 min at 95°C, vortexed, and then sonicated on a Diagenode Bioruptor Sonicator for 10 cycles (30 s on and 30 s off). Protein concentration was determined by a Pierce assay, and equal amounts of protein are loaded on Bio-Rad Mini-PROTEAN TGX gels 4 to 15%. After transfer on nitrocellulose, the membrane was blocked with Skin Milk Powder (5%) (Sigma-Aldrich) and exposed to primary antibodies overnight at 4°C [anti–α-tubulin 1/1000 (Sigma-Aldrich, T5168), anti–CTD 8WG16 1/1000 (Eurogentec, MMS-126P-050), anti–CTD Ser2P 3E10 1/1000 (Millipore, 04-1571), and anti-FLAG 1/1000 (Sigma-Aldrich, F3165)], washed three times in phosphate-buffered saline (PBS)–Tween (PBS-T; 0.05%), and then exposed to secondary antibody [anti-mouse immunoglobulin G (IgG) Perox (1/5000; GE, NA931) and anti-rat IgG Perox (1/5000; Dako, P0450)] and washed twice with PBS-T (0.05%). PerkinElmer Western Lightning Plus-ECL (enhanced chemiluminesence) was used for ECL and detection occurred on a GE ImageQuant LAS 4000 machine.

For Western blot on L1, L1s were grown for 4 hours and collected, washed once in M9, and frozen. Thawed worms were resuspended in 4× Laemmli Buffer [200 mM tris (pH 8.5), 8% SDS, and 40% glycerol] and then boiled for 5 min before being thoroughly vortexed and sonicated for 10 cycles (30 s on and 30 s off) on a Diagenode Bioruptor Sonicator. Protein concentration was measured by Bradford (Bio-Rad), and 15 μg of total protein was loaded on a Bio-Rad Mini-PROTEAN TGX gel 4 to 15%. Proteins were transferred to a nitrocellulose membrane using the Bio-Rad Trans-Blot Turbo transfer system, set up for high–molecular weight proteins (1.3A, up to 25 V). The membrane was then blocked in LI-COR Odyssey Blocking Buffer for at least 1 hour. Primary antibodies were incubated overnight at 4°C: anti–CTD S2P 3E10 1/1000 (Merck Millipore, 04-1571), anti–CTD S5P 3E8 1/1000 (ChromoTek), and anti–AMA-1 1/1000 (Novus Biologicals, 38520002). The membrane was washed three times in PBS-T and incubated for 1 hour with secondary antibodies coupled to infrared fluorophores (IRDye 800CW Goat anti-rat IgG, IRDye 800CW goat anti-mouse, and IRDye 680RD goat anti-rabbit) and then washed again two more times in PBS-T and one last time in PBS. Last, the membranes were dried out at room temperature, in the dark, overnight before the fluorescence was measured using the LI-COR Odyssey scanner and quantified using Image Studio (54).