In one experiment, a cDNA clone that contained a bp sequence from the coding region was used a control.Both probes detected the same pattern of transcript distribution.The RNA probes were generated using JCI UTP and T or T RNA polymerases essentially as described by the manufacturer. RNA probes were purified by ethanol precipitation in the presence of ammonium acetate, dissolved in formamide, mM DTT and stored at C.Sections were hybridized overnight in a humid chamber at gcc TAC tyr CAT his CTG leu GTG val TCA ser GGA gly AAG lys TCC ser TTT phe GCG ala tec CTC leu GAA glu GTG val CCG pro GAG glu GAG glu CCA pro CGG arg GTC val AGG arg GGA CAC his GTG val GAC asp CTG leu AGC ser ATG met GAA glu TTT phe CAA gin CAG gin TCC CAT his ATC lie ATC lie ATG net AAC asn AGC ser? CTG leu GGA gly CGT arg TTC phe GAG glu CAA gin AAG lys CGA arg CGG arg GAC asp CCG pro GCT ala GAG glu CCA pro AAG lys TGC cys AGC ser AAA lys AAG lys AGA arg TCG ser AGG arg TTA leu CAG gin ATT ile CCG pro GTG val CAG gin GAC asp CGC arg AAG lys CGT arg CGG arg CTG leu AAG lys GAG glu TCC ser CCC pro CAC his AGG arg AAG lys CAT his TGC cys CTG leu TCC ser ACC thr TGT cys ACG thr ATA ile ACA thr AAA lys TTG leu AAG lys TGAATTC C.Depending on the size of the coverslip, approximately J buffersection was used.Slides were washed in xSSC, formamide overnight and dried at room temperature.The nucleotide sequence of the primers for the amplification reaction was derived from the human cDNA sequence. The codons for the translational start and stop signals were included in order to obtain the complete coding region.Three different products were obtained, a bp VEGF, a bp VEGF and a bp VEGF cDNA. In contrast to the VEGF and VEGF PCR products, the VEGF cDNA was of very low abundance.Nucleotide sequences presented in lower case were obtained from a genomic DNA clone and differ at the indicated positions from the sequence of the PCR primer.The crosshatched box at the amino terminus represents the putative amino acid signal peptide for secretion.Additional domains in VEGF or VEGF are represented as shaded boxes.The direct protein sequence Atenolol analysis of the aminoterminal end of the putative murine VEGF is in agreement with the sequence obtained from the cDNA clones.However, the amino acid sequence differs at position which might be due to strain polymorphism.The inserted protein domain in the long VEGF form is highly basic, of amino acid residues being lysine or arginine.Interestingly, this exon is present only in an alternatively spliced mRNA variant and encodes a nuclear targeting signal. VEGF proteins of different mammalian species are highly Cabozantinib homologous with the rat VEGF protein.The amino acid sequence identities between the mature murine VEGF and the corresponding human and bovine proteins were each.The aminoterminal sequences of the mature proteins showed a considerable degree of sequence diversity.The resulting plasmids were then transfected into COS cells.Culture supernatants and extracts prepared from the transfected cells were assayed by immunoblot analysis for the presence of VEGF protein.