Rootletin

Antibiotics: ampicillin (Carl Roth, Karlsruhe, Germany), penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and puromycin (Thermo Fisher Scientific, USA).
Antibodies: ARL13B (1:100; Proteintech, Munich, Germany), GAPDH (1:10,000; Cell Signaling, Danvers, MA, USA), GT335 (1:1500; Proteintech, Germany), IFT88 (1:200; Proteintech, Germany), IFT140 (1:200; Proteintech, Germany), Rootletin (1:250; SantaCruz, Heidelberg, Germany), TTC30 (1:100; SantaCruz, Germany), acetylated tubulin (1:2500; Sigma, Burlington, MA, USA), and γ-tubulin (1:500; Novus Biologicals, Littleton, CO, USA); secondary antibodies: Alexa Fluor 488/568 (1:350; Invitrogen, Waltham, MA, USA) and goat α rabbit/mouse antibodies (1:10,000; Jackson ImmunoResearch, Philadelphia, PA, USA).
Cell culture: Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, USA) and fetal bovine serum (Sigma-Aldrich, USA).
Immunoprecipitation: anti-Flag-M2-agarose beads (Sigma-Aldrich, USA) and Flag-peptide (Sigma-Aldrich, USA).
Immunofluorescence staining: PFA (Morphisto, Offenbach, Germany) and Fluoromount-G (Invitrogen, USA).
Cell lines: HEK293T (CRL-3216, ATCC) for immunoprecipitation and protein complex analysis; hTERT-RPE1 cells (CRL-4000, ATCC) efficiently assemble primary cilia suitable for localization studies and phenotype analysis.

Cells were grown on coverslips and fixed with ice-cold methanol at -20°C for 10 min. The coverslips were then blocked with %5 BSA (Sigma; A3733) and primary antibodies were diluted in %1 BSA and incubated overnight at 4°C. The secondary antibody incubations were carried out at room-temperature for 1 hour, and washes were performed with PBS. Then coverslips were dried and then mounted on glass slides using a mounting medium containing DAPI (Vector Laboratories; H-1000-10).
The primary antibodies and their concentrations used in the experiments were: Centrin-3 (Abnova; H00001070-M01) at1/500 dilution; $\mathrm{\gamma }$ -tubulin (Sigma; T6557) at 1/500 dilution; $\mathrm{\gamma }$ -tubulin (Sigma; T3559): 1/250; CEP120 (Atlas Antibodies; HPA028823): 1/500; CEP152 (Bethyl Labs; A302-479A) at 1/500 dilution; CEP164 (Proteintech; 22227-1-AP) at 1/500 dilution; CEP170 (Bethyl Labs; A301-024A) at 1/250 dilution, C-Nap1 (Millipore; MABT1353) at 1/500 dilution; CEP68 (Proteintech; 15147-1-AP) at 1/500 dilution; Rootletin (Santa Cruz Biotechnology; sc-374056) at 1/500 dilution; Nek2 (BD; 610593) at 1/500 dilution. The secondary antibodies used in the experiments were: anti-rabbit AF488 (Invitrogen; A-11008), anti-rabbit AF555 (Invitrogen; A-31572), anti-mouse AF594 (Abcam; ab150116) and anti-mouse AF647 (Abcam; ab150115).