Reversal of Physiological Deficits Caused by Diminished Levels of Peptidylglycine {alpha}-Amidating Monooxygenase by Dietary Copper -- Bousquet-Moore et al. 150 (4): 1739 Data Supplement

Reversal of Physiological Deficits Caused by Diminished Levels of Peptidylglycine ${alpha}$ -Amidating Monooxygenase by Dietary Copper
Endocrinology Bousquet-Moore et al. 150: 1739

Supplemental Data

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Supplemental Data - Supplemental Data
Supplemental Tables 1 and 2 - Supplemental Tables 1 and 2
Supplemental Figure 1 - Amidation of TRH in Hypothyroid Mice. Immunoreactive TRH-Gly and TRH were quantified in extracts of PVN prepared from hypothyroid and euthyroid PAM^+/- (n=3-4) and WT male mice (n= 4-5). Error bars indicate SEM. Univariate statistical analysis revealed a significant difference separating the genotypes (p
Supplemental Figure 2 - : Identification of transcript generated from PAM^KO allele. (a) For genotyping, DNA was amplified using sense primers in Exon 3 and the neomycin resistance gene and an antisense primer in the intron separating Exons 3 and 4; WT mice yielded a 200 bp fragment while PAM^+/- mice yielded an additional 400 bp fragment. (b) cDNA prepared from the pituitaries of WT and PAM^+/- mice was amplified using a sense primer in Exon 3 and an antisense primer in Exon 6; consistent with the levels of PAM protein and PHM activity observed, the 300 bp product was more prevalent in WT mice than in PAM^+/- mice. (c) Amplification of PAM^+/- cDNA, but not WT cDNA with primers in the Neomycin resistance gene and Exon 6 yielded a 700 bp product that was sequenced. (d) Diagram shows the initial 6 exons in the PAM gene and the primers used.
Supplemental Figure 3 - Use of Laser Doppler to Monitor Vasoconstriction. PAM^+/- (N= 8) and WT (N = 8) mice were anesthetized with isoflurane and a laser Doppler probe was used to monitor blood flow through the tail vein before and for 5 min after an ip injection of phenylephrine (2 mg/kg). Averaged data for each time point are expressed as a percentage of baseline blood flow prior to phenylephrine injection; error bars, SEM. Both PAM^+/- and WT mice showed similar levels of pharmacologically-induced vasoconstriction in response to phenylephrine.