pecfp n1 expression vector  (TaKaRa)

Journal: The Journal of Biological Chemistry

Article Title: The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering *

doi: 10.1074/jbc.M112.349209

Figure Lengend Snippet: Binding of exogenous fl-HA by COS-7 cells transfected with CD44 expression constructs. 24 h post-transfection, COS-7 cells were analyzed by Western blot and flow cytometry analysis. The capacity of transfected cells to bind exogenous fl-HA was analyzed by flow cytometry and immunocytochemistry analysis. A , shown is immunoblotting of CD44 in the lysates from CD44-CFP ( lane 2 )- and CD44-YFP ( lane 3 )-transfected COS-7 cells. Naive COS-7 cell was used as the negative control. B , cell surface expression of CD44 in CD44-CFP- and CD44-YFP-transfected COS-7 cells was assessed by flow cytometry. Histograms indicate log fluorescence intensity ( x axis) versus relative cell number ( y axis). 1 , naïve COS-7 cells was used as mock; 2 , isotype-matched antibody as negative control; 3 , pECFP-N1 transfected cells; 4 , pEYFP-N1 transfected cells; 5 , CD44-CFP transfected cells; 6 , CD44-YFP transfected cells. C , shown is binding and uptake of exogenous fl-HA by transfected COS-7 cells. 1 , naïve COS-7 cells was used as negative control; 2 , CD44-CFP transfected COS-7 cells; 3 , CD44-YFP transfected COS-7 cells. D , binding of exogenous fl-HA by transfected COS-7 cells was detected by immunocytochemistry analysis. Nuclei were visualized by propidium iodide ( PI ) staining. COS-7 cells were transfected with CD44-CFP or CD44-YFP respectively. Naïve COS-7 cells were used as mock control.

Article Snippet: The cDNA encoding the hematopoietic form of CD44 (CD44-H) was obtained by reverse transcription PCR (RT-PCR) using total RNA isolated from human peripheral blood lymphocytes and subcloned into the mammalian expression vectors pECFP-N1 and pEYFP-N1 (Clontech, Palo Alto, CA).

Techniques: Binding Assay, Transfection, Expressing, Construct, Western Blot, Flow Cytometry, Immunocytochemistry, Negative Control, Fluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering *

doi: 10.1074/jbc.M112.349209

Figure Lengend Snippet: nHA and oHA regulate CD44 clustering in COS-7 cells transfected with CD44 expression constructs. Clustering of CD44-CFP and CD44-YFP was detected by the efficiency of FRET between donor (CFP) and acceptor (YFP). Fluorescence at 485 nm (CFP emission peak) and 528 nm (YFP emission peak) were measured after excitation only at 440 nm (CFP special excitation channel). The ratio of fluorescence at 528 and 485 was estimated to indicate the transferred energy of CFP to YFP. CD44-CFP , transfected with CD44-CFP plasmid; CD44-YFP , transfected with CD44-YFP; CFP , transfected with pECFP-N1 vector; YFP , transfected with pEYFP-N1 vector; PMT-CFP-YFP , CFP-YFP fusion protein with a plasma membrane target (PMT) sequence at the CFP N terminus. A , fluorescence spectra of recombinant CFP and YFP proteins are shown. B , nHA and oHA treatment led to the changes in the efficiency of energy transfer from cyan to yellow fluorescent proteins in COS-7 cells co-transfected with CD44-CFP and CD44-YFP. C , oHA abrogated CD44 clustering triggered by exogenous nHA. After exposed to nHA, cells were treated with oHA of different concentrations (0–250 μg/ml) for another 2 h. The fluorescence intensity was plotted as the mean ( n = 8) ±S.D. RFU , relative fluorescence units.

Article Snippet: The cDNA encoding the hematopoietic form of CD44 (CD44-H) was obtained by reverse transcription PCR (RT-PCR) using total RNA isolated from human peripheral blood lymphocytes and subcloned into the mammalian expression vectors pECFP-N1 and pEYFP-N1 (Clontech, Palo Alto, CA).

Techniques: Transfection, Expressing, Construct, Fluorescence, Plasmid Preparation, Sequencing, Recombinant