Cilia Protein Detection by Western Blot

Isolated cilia were denatured in LDS-sample buffer (Boston BioProducts) for 10 min at 95C. The denatured samples were then loaded onto a precast 4–20% SurePAGE Bis-Tris gel (GenScript) and separated by electrophoresis in Tris-MOPS-SDS running buffer (GenScript) before being transferred to nitrocellulose membranes (Cytiva). Membranes were blocked with 5% skim milk in TBST (Tris-buffered saline with Tween-20), followed by addition of primary antibodies (Key Resources Table). Antibodies against PKD2-Cterm^{48 (link)} and β-tubulin (Cell Signaling Technology, RRID: AB_2210545) were used at a 1:1000 dilution. Antibodies against PKD2-loop, ^{48 (link)} MST1,^{19 (link)} and SIP^{19 (link)} were used at 1:2000 dilution. Primary antibodies were detected with a horseradish peroxidase-conjugated anti-rabbit antibody (Sigma-Aldrich, RRID: AB_92591) at 1:2000 dilution. After washing in TBST, peroxidase substrate (Cytiva) was applied to the membrane and imaged using a Gel Doc XR+ imaging system and the Image Lab software (Bio-Rad).