Figure 7

PTB antibodies precipitate Cx55,5 mRNA from transiently transfected N2A cells: Binding of PTB to Cx55.5 in living cells was tested 36 hrs post transfection. Crosslinked cell lysates were subjected to immunoprecipitate Cx55.5 RNAs using anti-PTB or anti-β-actin antibodies. RNAs binding to the antibody captured proteins were heat released, reversed transcribed and PCR amplified using primers specific for the Cx55.5 coding region. Lane I) NTC, no template control, lane II) control, PCR of cDNA reactions obtained from the samples immunoprecipitated with Protein A sepharose beads only (without any antibody), III) PCR reaction of cDNA obtained from the RNA immunoprecipitated from anti β-actin coated beads, IV) PCR of cDNA obtained from RNA reverse transcribed with primer pairs specific for the entire coding region (1497). V) cDNA from RNA reverse transcribed with nested primers for the carboxy-terminus fragment (400 bp). VI) RT-minus sample; RNA immunoprecipitated with PTB antibodies without reverse transcription.