Expression of AmAOX transcripts and proteins in the appendices of A. maculatum.
(a) Infrared thermal images of the pre-hot (β-stage), non-hot (γ/δ-stage) and hot (δ-stage) appendices used in this study. (b) AmAOX transcript levels in pre-hot, non-hot and hot stages of A. maculatum. These levels are shown as an expression ratio relative to EF1a. Expression levels analyzed by qRT-PCR and cycleave PCR are shown at the bottom of the figure. The total AmAOX expression level was determined using common primers that amplify all types of transcripts (AmAOX1a–AmAOX1g; denoted by ‘all’). Transcripts for QNT- and QDT-type AmAOXs were coamplified in the cycleave PCR (QNT + QDT). Values are the means ± SD (n = 3). Different alphabetical letters in the graph indicate significantly different values (P < 0.05). (c) Expression of AmAOX proteins in the mitochondria from various thermogenic stage appendices. Mitochondrial proteins were separated by SDS–PAGE, visualised with the Coomassie Brilliant Blue (CBB) staining (right), transferred to a polyvinylidene difluoride membrane and immunoblotted with antibodies against AOX (left). Molecular mass standards are indicated.