Cells of JSCA0021 were plated with 5 FOA to induce recombination between two copies of dpl200 flanking the mini Ura blaster for a loss of CaURA3 to generate JSCA0022. To allow the expression of cassettes encoding assorted CaCdc4 domains in selleck Ponatinib C. albicans, a Tet on plasmid, pTET25M, which is derived from pTET25 for inducing gene expression with Dox, has been developed. To regulate CaCDC4 expression by the Tet on system, the coding sequence of CaCDC4 was PCR amplified using plasmid CaCDC4 SBTA bearing CaCDC4, primers CaCDC4 SalI and CaCDC4 BglII, and Pfu polymerase, digested with SalI and BglII for cloning into pTET25M, from which pTET25M CaCDC4 was gener ated. Moreover, CaCDC4 6HF, which encodes 6��histi dine and FLAG tags at the C terminal of CaCdc4, was PCR amplified with primers CaCDC4 6HF SalI and CaCDC4 6HF BglII, followed by digestion with SalI and BglII and cloning into pTET25M to obtain pTET25M CaCDC4 6HF.

To define the function of the distinct CaCdc4 domains, different CaCDC4 portions were used to replace the full length CaCDC4 coding sequence on pTET25M CaCDC4 6HF. By using the primer sets listed in Table 2, the following constructs were made, pTET25M NCaCDC4 6HF, which encodes the N terminal truncated CaCdc4, pTET25M F 6HF, which encodes the F box domain with flanking regions, pTET25M WD40 6HF, which encodes eight copies of WD40 repeat, and pTET25M NF 6HF, which encodes truncated N terminal CaCdc4 and the F box domain. All inserts of the constructs were released with AatII and XhoI to replace the full length CaCDC4 on pTET25M CaCDC4 6HF.

Consequently, plasmids bearing those CaCDC4 segments flanked with common C. albicans ADH1 sites were digested with SacII and KpnI, each of which was transformed into C. albicans for integration at the CaADH1 locus. All strains were verified by colony PCR with specific primers before subjecting to Southern blotting analysis. Southern blotting analysis Genomic DNA from the C. albicans strains was isolated by the MasterPure Yeast DNA Purification Kit according to the manu factures instruction. Southern blotting was performed with the aid of the Rapid Downward Transfer System using 10 ug of the restriction enzyme digested genomic DNA. The DNA on the blot was hybridized with a probe amplified by the PCR DIG probe synthesis kit with the primers CaCDC4 Probe F and CaCDC4 Probe R for CaCDC4 locus or CaADH1 Probe F and CaADH1 probe R for ADH1 locus using DIG Easy Hyb.

To reveal the structure of gene locus, the DIG Luminescent Detection Kit was used after hybridization, and the luminescent images of blot were captured with the imaging analysis system. Protein extraction and Western blot analysis Cultured cells were collected, and the Entinostat total protein from each sample was extracted as described previously. The proteins were resolved by 10% SDS PAGE and transferred to PVDF membranes.