Fig. 4.
Fig. 4. Quantitation of CD40L mRNA transcripts by RNase protection assay. (A) Design of probes used in the RNase protection assay (RPA). The upper open bar shows the domain structure of the CD40L molecule; IC, intracellular tail; TM, transmembrane domain; ECU, extracellular unique region; and TNFH, TNF-homology domain. The numbers above the upper open bars indicate the starting amino acid residue for each domain. The lower open bar shows the contribution of each exon to the CD40L domains. The three closed bars indicate the locations and nucleotide borders of the RPA probes used. (B) Autoradiograms of representative RPAs using probes A, B, and C. In each autoradiogram, arrowheads indicate the protected bands derived from CD40L mRNA transcripts and horizontal bars indicate those derived from CD3δ mRNA transcripts. Total RNA was isolated from activated PBMC of normal controls in lanes 1, 3, and 5, and selected XHIM patients: JG (family 28) in lane 2, PS (family 18) in lane 4, and AM (family 19) in lane 6. Radioactivity of protected CD40L and CD3δ mRNA transcripts was quantitated by the PhosphorImager analysis system and the ratio of CD40L/CD3δ mRNA transcripts calculated as follows: lane 1, 0.883; lane 2, 0.205; lane 3, 1.021; lane 4, total 1.072 (0.692 for 275 bp and 0.380 for 178 bp); lane 5, 0.837; and lane 6, total 0.838 (0.290 for 205 bp and 0.548 for 147 bp). (C) Quantitation of CD40L mRNA transcripts isolated from activated PBMC of XHIM patients. The ratio of CD40L/CD3δ mRNA transcripts was plotted on the ordinate. The number attached to each symbol (representing an individual XHIM patient) corresponds to the patient's family number (see Table 1). In patients with intron 2 splice site mutations (families 17 and 18), mRNA transcripts in which exon 2 was skipped (▿) or 19 nucleotides were inserted (▵) were quantitated separately using probe B; the total amount of transcripts is indicated by an open diamond (◊). In patients with intron 3 splice site mutations (families 19 and 20), normally spliced (▾) and exon 3-skipped (▴) mRNA transcripts were quantitated separately using probe C; the total amount is indicated by a closed diamond (⧫).

Quantitation of CD40L mRNA transcripts by RNase protection assay. (A) Design of probes used in the RNase protection assay (RPA). The upper open bar shows the domain structure of the CD40L molecule; IC, intracellular tail; TM, transmembrane domain; ECU, extracellular unique region; and TNFH, TNF-homology domain. The numbers above the upper open bars indicate the starting amino acid residue for each domain. The lower open bar shows the contribution of each exon to the CD40L domains. The three closed bars indicate the locations and nucleotide borders of the RPA probes used. (B) Autoradiograms of representative RPAs using probes A, B, and C. In each autoradiogram, arrowheads indicate the protected bands derived from CD40L mRNA transcripts and horizontal bars indicate those derived from CD3δ mRNA transcripts. Total RNA was isolated from activated PBMC of normal controls in lanes 1, 3, and 5, and selected XHIM patients: JG (family 28) in lane 2, PS (family 18) in lane 4, and AM (family 19) in lane 6. Radioactivity of protected CD40L and CD3δ mRNA transcripts was quantitated by the PhosphorImager analysis system and the ratio of CD40L/CD3δ mRNA transcripts calculated as follows: lane 1, 0.883; lane 2, 0.205; lane 3, 1.021; lane 4, total 1.072 (0.692 for 275 bp and 0.380 for 178 bp); lane 5, 0.837; and lane 6, total 0.838 (0.290 for 205 bp and 0.548 for 147 bp). (C) Quantitation of CD40L mRNA transcripts isolated from activated PBMC of XHIM patients. The ratio of CD40L/CD3δ mRNA transcripts was plotted on the ordinate. The number attached to each symbol (representing an individual XHIM patient) corresponds to the patient's family number (see Table 1). In patients with intron 2 splice site mutations (families 17 and 18), mRNA transcripts in which exon 2 was skipped (▿) or 19 nucleotides were inserted (▵) were quantitated separately using probe B; the total amount of transcripts is indicated by an open diamond (◊). In patients with intron 3 splice site mutations (families 19 and 20), normally spliced (▾) and exon 3-skipped (▴) mRNA transcripts were quantitated separately using probe C; the total amount is indicated by a closed diamond (⧫).

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