Ift140

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.

Antibodies against acetylated α-tubulin, γ-tubulin, Flag (Sigma-Aldrich), acetylated lysine, α-tubulin, Cep164, ninein (Santa Cruz Biotechnology), TMEM67, MKS1, IFT140, IFT88, CP110 (Proteintech), HDAC6 (abgent), and GFP (Roche) were purchased from the indicated sources. Rhodamine- and fluorescein-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, and Alexa Fluor 405-conjugated secondary antibodies were from Invitrogen. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Tubastatin A and tubacin were obtained from Santa Cruz Biotechnology. DAPI and tetramethylrhodamine-conjugated phalloidin were from Sigma-Aldrich. Control and HDAC6 siRNAs and plasmids expressing HA-HDAC6, GFP-HDAC6, and GFP-α-tubulin were described previously38 (link)44 (link), and various mutants were generated by PCR and site directed mutagenesis. The plasmid expressing mCherry-β-tubulin was constructed by PCR using the pmCherry-C1 vector (Clontech). Plasmids expressing Flag-cortactin wild-type, 9KQ, and 9KR plasmids were obtained from Edward Seto (H. Lee Moffitt Cancer Center & Research Institute, Tampa, USA).