Pcdh ef1 mcs ires puro

Full-length cDNAs of human CCT1-8 subunits of the CCT were generous gifts from Dr. Jiahuai Han at Xiamen University, China. The coding regions of these genes were cloned into pcDNA3.3 (Thermo Fisher Scientific) with an N-terminal Flag tag. Mouse WDTC1 were cloned from a cDNA library prepared from testis mRNA of a C57BL6 mouse. A pIRESpuro-GLUE vector (Addgene, #15100) (Angers et al., 2006 (link)) was used to generate a stable cell line for the purification of WDTC1-interacting proteins. For lentiviral overexpression, genes were cloned into either pCDH-EF1-MCS-IRES-Puro (System Biosciences) or pCDH-EF1-MCS-IRES-BSD (modified from the former one by replacing the puromycin resistance gene to blasticidin resistant gene) with or without the N-terminal Flag tag. For knockdown of genes, a pLKO.1 vector (Addgene, 10878) (Moffat et al., 2006 (link)) was used. The primer sequences are listed in Supplementary Table S1.

Full-length cDNA of human ARF1, ARF3, ARF4, ARF5, ARF6, EXOC1-8, EFA6B, and EGFR were cloned from a cDNA library prepared from HEK293T or HeLa cells. These genes were cloned into either pcDNA3.3 or pCDH-EF1-MCS-IRES-Puro (pCDH-puro, System Biosciences) with indicated tags. For knockdown, shRNAs were cloned into pLKO.1 (Addgene, 10878) or pLL3.7 (Addgene, 11795). SgRNA was cloned into pLentiCRISPR (Addgene, 52961). Site-directed mutagenesis was performed using commercial kits from New England Biolabs. The primer sequences are listed in Supplementary Table 1.

Mouse Glis2 (NCBI Accession NM_031184) was cloned into several vectors with different epitope tag combinations: pLenti CMV GFP Blast (Addgene, Cat. no. #17445) with N-terminal V5 tag and C-terminal EGFP tag; pLVX-TetOne (Takara, Cat. no. 631846) with C-term FLAG tag; pHTC HaloTag® CMV-neo was used to clone Glis2 with C-term Halo tag. Human GLIS2 (NCBI Accession NM_032575) was purchased as GLIS2-HaloTag® human ORF in pFN21A (Promega, Cat. no. FHC03277). Mouse Glis3 (NCBI Accession NM_175459) was cloned into pLenti CMV GFP Blast with C-term EGFP tag. The cilia marker Nphp3^(1-200)-mApple was made by amplifying a fragment corresponding to amino acid 1-200 of mouse Nphp3 (NCBI Accession NM_028721.3) from mouse kidney cDNA and combining it in-frame with mApple using PCR, followed by subcloning into a pCDH-Hygro vector which was modified from pCDH-EF1-MCS-IRES-Puro (System Biosciences). All constructs were validated by sequencing and immunoblot expression analysis. Transient transfection of HEK293T cells was done with Lipofectamine 2000 (Thermo Fisher Scientific, Cat. no. 11668030). Stable gene expression was achieved using lentivirus transduction and selection with appropriate antibiotic.