GATA1 mutations affect the expression level of the truncated protein. (A) The expression levels of GATA1s protein and mRNA were assessed in BHK-21 cells transfected with human GATA1 minigene expression vectors carrying mutations observed in TAM patients. Western blot analysis was performed with anti-GATA1 (i) or anti-NeoR antibody (ii). Northern blot analysis was carried out with GATA1 exon 3-6 fragment (iii) or NeoR cDNA (iv) as probe. (B) The expression levels of GATA1s protein and mRNA in BHK-21 cells transfected with human GATA1 minigene expression vectors with PTC type 1 mutation. Levels were assessed by Western blot analysis with anti-GATA1 antibody (i), anti-NeoR antibody (ii). Northern blot analysis was performed with GATA1 exon 3-6 (iii), exon 2 (iv), or NeoR cDNA (v). To detect the transcripts derived from the human GATA1 minigene expression construct, RT-PCR analysis was carried out using primers described in “RT-PCR” (vi). Ex 2(+) and Ex 2(−) indicate PCR products or transcripts with or without exon 2, respectively. Ratio of Ex 2(−)/(+) was calculated from the results of a densitometric analysis of the RT-PCR. The asterisk denotes unavailable data (vii).