摘要
Purpose:To study the expression of four plasma membrane calcium ATPase (PMCA) isoforms in human lens epithelium cell lines (HLE-B3 cells) both on mRNA and protein levels.Methods:Both total mRNA and membrane protein samples were collected,after HLE-B3 cells were cultured to 90% confluency.Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to detect mRNAs of PMCA isoform 1,2,3,and 4 by using corresponding PMCA isoform 1,2,3,and 4 primers.Western Blot analysis was employed to detect PMCA isoform 1,2,3,and 4 protein using corresponding anti-PMCA1,2,3,and 4 antibodies.Results:A 420 bp fragment was amplified with PMCA1 primer.A 550 bp fragment was amplified with PMCA2 primer.A 840 bp fragment was amplified with PMCA4 primer.No fragment was amplified with PMCA3 primer.Western Blotting confirmed that the expected ～153 kDa,～125 kDa and ～147 kDa protein were recognized by anti PMCA1,2 and 4 antibodies respectively.No protein was recognized by PMCA3 antibody.Conclusion:This is the first study showing only PMCA1,2,and 4 gene are expressed in HLE-B3 cells on both mRNA and protein level.PMCA3 is not expressed in HLE-B3 cells.The PMCA isoforms expression pattern in HLE-B3 cell lines is different from that in the lens of other species.PMCA2 may play a more important role over other isoforms.
Purpose：To study the expression of four plasma membrane calcium ATPase （PMCA） isoforms in human lens epithelium cell lines （HLE-B3 cells） both on mRNA and protein levels.Methods：Both total mRNA and membrane protein samples were collected,after HLE-B3 cells were cultured to 90% confluency.Reverse Transcription Polymerase Chain Reaction （RT-PCR） were used to detect mRNAs of PMCA isoform 1,2,3,and 4 by using corresponding PMCA isoform 1,2,3,and 4 primers.Western Blot analysis was employed to detect PMCA isoform 1,2,3,and 4 protein using corresponding anti-PMCA1,2,3,and 4 antibodies.Results：A 420 bp fragment was amplified with PMCA1 primer.A 550 bp fragment was amplified with PMCA2 primer.A 840 bp fragment was amplified with PMCA4 primer.No fragment was amplified with PMCA3 primer.Western Blotting confirmed that the expected ～153 kDa,～125 kDa and ～147 kDa protein were recognized by anti PMCA1,2 and 4 antibodies respectively.No protein was recognized by PMCA3 antibody.Conclusion：This is the first study showing only PMCA1,2,and 4 gene are expressed in HLE-B3 cells on both mRNA and protein level.PMCA3 is not expressed in HLE-B3 cells.The PMCA isoforms expression pattern in HLE-B3 cell lines is different from that in the lens of other species.PMCA2 may play a more important role over other isoforms.
