眼科学报（英文版）
眼科學報（英文版）
안과학보（영문판）
EYE SCIENCE
2011年
1期
35-43
,共9页
plasma membrane calcium ATPase%gene expression%reverse transcription-polymerase chain reaction (RTPCR)%HLE-B3 cells%Western Blot
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 isoforrn 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 PMCAl 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.
