Anti ha tag c29f4 rabbit mab

30 μg of total protein for input or 45 μl of pulldown supernatant was loaded into 4–20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels. Total protein was assessed through stain-free imaging on a Biorad ChemiTouch Imaging System, which allows total protein loaded into the well to be used as the loading control. Protein was transferred onto the PVDF membrane (Biorad, 162–0255) using the Trans-Blot Turbo Transfer System (Biorad 1704150). Membranes were blocked with 5% milk in 0.1% TBST for 1 hour and probed with one of the following antibodies at the designated dilution overnight at 4°C. 1:3000 Monoclonal ANTI-FLAG® M2 antibody (Sigma, F1804, Lot. SLBK1346V), 1:1000 anti-His HRP antibody (GenScript, A00612, Lot. 19K001984), 1:1000 anti-HA-Tag (C29F4) Rabbit mAb (Cell Signaling, 37245, Lot. 8), or 1:1000 anti-Human/Mouse/Rat PRL-3 Antibody (R&D Systems, MAB3219, Lot. WXH0419091). Following three washes with 0.1% TBST, secondary HRP-conjugated 1:2500 anti-mouse IgG antibody (Cell Signaling, 7076S, Lot. 33) or 1:5000 anti-Rabbit IgG HRP Linked F(ab′)2 (Sigma, NA9340V, Lot. 17065618) was added for 1 hour and membranes were imaged using Clarity Western ECL Substrate (Biorad, 1705061).

Synchronized young adult animals expressing N-terminal HA-tagged PMK-1 were used for immunoprecipitation with anti-HA-Tag (C29F4) rabbit mAb (Cell Signaling Technology 11846) and eluted with 2× laemmli buffer. Eluted protein was resolved by SDS-PAGE and gel slices of PMK-1 band were sent for protein ID using Mass Spectrometry. Trypsinization, desalting, mass-spectrometry and analyses were performed by the UTSW proteomics core. For phospho-peptide analysis, samples were run on an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Data analysis was performed using Proteome Discoverer 2.4 SP1 using the C. elegans protein database from UniProt. The ratio of the abundance of T196 phospho-peptide to the unphosphorylated peptide was used to estimate the percentage of phosphorylated PMK-1.