Monoclonal anti p27 antibody

The CDKN1B mutation identified in the proband by sequencing was introduced by site-directed mutagenesis (Quikchange II Site-Directed Mutagenesis Kit, Stratagene, La Jolla, CA, USA) into the full-length WT human CDKN1B cDNA cloned in a pEYFP (EYFP, enhanced yellow fluorescent protein) plasmid backbone. The mutant protein was expressed in human cervix adenocarcinoma (HeLa) and rat pituitary epithelial-like tumor (GH3) cell lines, maintained in DMEM media supplemented with 10% fetal bovine serum, 20 mM l-glutamine, 100 units/ml of penicillin G sodium, and 100 μg/ml streptomycin, or F-12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum, 20 mM l-glutamine, 100 units/ml of penicillin G sodium, and 100 μg/ml streptomycin, respectively (Invitrogen). Transient transfections were carried out as reported previously (7) (link). For western blotting and immunofluorescence analyses, the resulting vector expressed the mutant P27 protein as a fusion protein with the YFP tag at the N-terminus. Indirect immunofluorescence was performed using a monoclonal anti-P27 antibody (BD Biosciences, San Jose, CA, USA) as reported previously (7) (link).