Polyclonal anti gfp

MDCK cells were grown in DMEM (Sigma-Aldrich) containing 5% FBS. MDCK cells were cotransfected with sequences encoding for GFP-FR and PLAP or with mCherry-PLAP or mCherry-p75 (the latter two were cloned by PCR in pcDNA3.1 vector carrying hygromicin resistance). In selected experiments we used MDCK cells stably transfected with p75-GFP (kind gift of Dr. A. Le Bivic, IBDML, Marseille, France) or with mGFP-FR (cloned by PCR in PJB20 vector carrying neomicin resistance). Monomeric GFP (mGFP) was derived by site-directed mutagenesis replacing the hydrophobic amino acids (in the external part of the β-barrel) with positively charged amino acids^{43 (link)}. One, two or three mGFP in tandem were engineered and cloned by PCR in pcDNA3.1 vector. CHO cells stably expressing GFP-FR (kind gift of Dr. S. Mayor, NCBS Bangalore, India) were grown in HAM's F12 medium containing 10% FBS and were transiently co-transfected with mCherry-PLAP.
The cDNAs encoding for mGFP-GPI (uPAR) and mGFP-mGFP-GPI (uPAR), in which one or two mGFP, respectively, are fused to the GPI attachment signal of uPAR^{44 (link)}, were a kind gift of Dr. N. Sidenius (IFOM, Milan, Italy) and were transiently transfected in MDCK cells.
We used the following antibodies: polyclonal and mouse monoclonal anti-PLAP (Rockland and Sigma-Aldrich, respectively), polyclonal anti-GFP (Clontech).