HEK293T cells (American Type Culture Collection CRL-
3216) were grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum, 2 mMl -glutamine, penicillin (5 U/ml), and streptomycin (50 μg/ml). To introduce expression vectors, HEK293T cells were transfected with
PEI MAX (Polysciences Inc., Warrington, PA, USA). UFL1
(5′-CCAGCGGGCGCAGTTCGCCG-3′) or UFBP1 (5′-GTAGCGGCGGCTCTGCTAGT′) guide RNA was designed using the CRISPR Design tool (https://crispr.dbcls.jp/ ) and subcloned
into pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, #42230), a human codon–optimized SpCas9 and chimeric guide RNA expression plasmid. To generate UFL1, UFC1, CDK5RAP3, and UFBP1 UFSP2 KO HEK293T cells, HEK293T or UFSP2 KO HEK293T cells (16 (link)) were transfected the aforementioned pX330 vectors together with pEGFP-C1 (#6084-1, Clontech Laboratories, Mountain View, CA, USA) and cultured for 2 days. GFP-positive cells were sorted and expanded. Ablation of UFL1, UFC1, or CDK5RAP3 was confirmed by a heteroduplex mobility assay followed by immunoblot analysis with anti-UFL1, anti-UFC1, or anti-CDK5RAP3 antibody. UFM1- (23 (link)), UFC1- (23 (link)), UFBP1- (27 (link)), CDK5RAP3- (27 (link)), and UFSP2-deficient HEK293T cells (16 (link)) were used in this study. HEK293T and HeLa cells were authenticated by short-tandem repeat profile. All cell lines were tested for mycoplasma contamination.
3216) were grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum, 2 mM
PEI MAX (Polysciences Inc., Warrington, PA, USA). UFL1
(5′-CCAGCGGGCGCAGTTCGCCG-3′) or UFBP1 (5′-GTAGCGGCGGCTCTGCTAGT′) guide RNA was designed using the CRISPR Design tool (
into pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, #42230), a human codon–optimized SpCas9 and chimeric guide RNA expression plasmid. To generate UFL1, UFC1, CDK5RAP3, and UFBP1 UFSP2 KO HEK293T cells, HEK293T or UFSP2 KO HEK293T cells (16 (link)) were transfected the aforementioned pX330 vectors together with pEGFP-C1 (#6084-1, Clontech Laboratories, Mountain View, CA, USA) and cultured for 2 days. GFP-positive cells were sorted and expanded. Ablation of UFL1, UFC1, or CDK5RAP3 was confirmed by a heteroduplex mobility assay followed by immunoblot analysis with anti-UFL1, anti-UFC1, or anti-CDK5RAP3 antibody. UFM1- (23 (link)), UFC1- (23 (link)), UFBP1- (27 (link)), CDK5RAP3- (27 (link)), and UFSP2-deficient HEK293T cells (16 (link)) were used in this study. HEK293T and HeLa cells were authenticated by short-tandem repeat profile. All cell lines were tested for mycoplasma contamination.
