Multiimage 3

Tissue total lysates were prepared in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1% Triton X-100) and 20–30 μg of protein were separated by SDS PAGE electrophoresis and transferred to nitrocellulose membranes. Acot1 (ab133948, Abcam, Cambridge, MA), Acot7 (affinity purified antibody [41 (link)]), and Hsc70 (sc-59570, Santa Cruz, Dallas, TX) coupled with anti-rabbit Cy5 (Invitrogen, Grand Island, NY) or anti-mouse Cy3 (Invitrogen) were visualized with Alpha Innotech MultiImage III and quantified using Alpha Innotech FluorChem Q Software (Santa Clara, CA), and data was normalized to Hsc70 expression.

cDNA was synthesised with the GoScript reverse transcription system (Promega, UK). Total RNA was reverse transcribed using a 1:1 mixture of random primers and oligo(dT). The GoTaq hot start polymerase kit (Promega, UK) was used to perform standard PCR. PCR conditions were: initial denaturation at 94 °C for 2 min; then 35–40 cycles: 94 °C for 30 s; 58 °C for 30 s; and 72 °C for 30 s followed by a final extension at 72 °C for 5 min. Primer sequences used in standard PCR are shown in Additional file 1: Table S1. PCR products were run on 2% (w/v) agarose gels for 1 h at 95 V and analysed using FluorChem Q software, Alpha Innotech MultiImage III. Optical density peak values were generated with ImageJ software. Excel was used to calculate the percentage splicing index (PSI/ψ) where ψ = exon inclusion/ exon inclusion + exon skipping band intensities.