Lithium dodecyl sulfate (lds)

GST-NLRP3 (1 μg, Abcam) was incubated of with GST-CSNK1A1 (100 ng, Sino Biological), GST-CSNK2A1 (100 ng, Sino Biological)/GST-CSNK2B (100 ng, Creative Biomart), or 6-His-GST-CAMK2B (100 ng, Sino Biological)/Calmodulin (1 μg, Enzo) with 50 μM ATP and 4 μCi of [γ³²P]-ATP (PerkinElmer) in kinase buffer (50 mM HEPES pH 7.3, 100 mM NaCl, 10 mM MgCl₂, 0.05% Triton X-100, 10 mM β -glycerophosphate, 5 mM NaF) at 30 °C for 30 min in a final volume of 39 μL, supplemented with 2 mM CaCl₂ for CAMK2B/Calmodulin reactions. Reactions were stopped by adding 15 μL of LDS (ThermoFisher Scientific) and 6 μL of DTT (Euromedex) 500 mM. Samples were analyzed by electrophoresis using Bolt 4–12% Bis-Tris Plus gel (ThermoFisher Scientific) followed by Coomassie blue gel staining using PageBlue^TM Protein staining solution (ThermoFisher Scientific) and autoradiography.

Spinal cord homogenates were prepared in T-PER tissue protein extraction reagent (ThermoFisher Scientific) containing protease inhibitors (Roche). Protein concentration was measured by BCA assay (ThermoFisher Scientific). Protein equivalents from each sample were boiled in LDS (ThermoFisher Scientific) containing 2.5% β-mercaptoethanol and electrophoresed on 4–12% Bis-Tris acetate gels (BioRad). Protein was transferred to nitrocellulose (BioRad) in transfer buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, 0.1% (w/v) SDS, and 20% methanol. Membranes were blocked for 1 h in 20 mM Tris, 500 mM NaCl, pH 7.5 (TBS) containing 5% milk, then incubated overnight at 4 °C with primary antibodies diluted in TBS-0.1% tween-20 (TBST) containing 5% milk. After washing in TBST, membranes were incubated with HRP-conjugated goat-anti-rabbit or goat-anti-mouse antibodies (GE Life Sciences) and detected using ECL reagents (GE Life Sciences). Primary antibodies included rabbit anti-beta-galactosidase (1:500, MP/Cappel), mouse anti-beta-tubulin (1:1000, Sigma), rabbit anti-SLC7A10, N-term (1:1000, Acris, lot #FGI263), mouse anti-GLYR (1:1000, Synaptic Systems), rabbit anti-VIAAT (1:1000, Aviva), rabbit anti-GLYT1 (1:1000, Aviva), and mouse anti-GLYT2 (1:1000, Millipore). Densitometry was performed using ImageJ.

Cells were lysed with RIPA (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitors (cOmplete Mini, Roche) and phosphatase inhibitors (PhosSTOP). Lysates were clarified by centrifugation, and protein concentration of supernatants was evaluated using BCA (Pierce). Protein lysates were normalized, mixed with LDS (Thermo Fisher) and β-mercaptoethanol (Thermo Fisher), and denatured at 95°C for 5 min. Protein was separated by gel electrophoresis using 4–12% or 12% NuPAGE Bis-Tris gradient gels (Life Technologies) and transferred to nitrocellulose membranes (GE Healthcare, 0.45 μm). Membranes were blocked with 5% BSA in TBS supplemented with 0.1% Tween 20, and primary antibodies were stained overnight in blocking buffer at 4°C or 1 hr at room temperature, and HRP-conjugated secondary antibodies were stained for 1 hr at room temperature. Membranes were washed and then incubated with ECL plus chemiluminescent reagent (Pierce) for 30 s. Chemiluminescent signal was evaluated using CL-XPosure Film (Thermo Fisher).

Western blotting of ciliary proteins was performed using standard protocols, as previously described [7 (link),34 (link)]. For characterization of anti-ODAD1 antibodies, plasmids encoding the 670 amino acid isoform (Uniprot.org, accession # Q96M63) and the predicted 437 amino acid truncated isoform were constructed by GeneScript (GeneScript, Piscataway, NJ, USA). Both constructs contained an amino-terminal HA-tag. Plasmids were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and cell lysates were prepared in RIPA buffer. Cilia from differentiated cultures of airway epithelial cells were isolated as previously described [7 (link),34 (link)], pelleted, and resuspended in 1× LDS (Invitrogen, Waltham, MA, USA) lysis buffer. Proteins were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA), transferred to nitrocellulose membranes and blocked in OneBlock (Genesee Scientific, San Diego, CA, USA). Membranes were probed with the antibodies and concentrations listed in Table S3 and visualized using a LI-COR Odyssey Scanner (LI-COR Biotechnology, Lincoln, NE, USA).