Two hundred and fifty milligrams of fully expanded leaf tissue was snap frozen in liquid N2 and was stored at −80 °C. The frozen tissue was ground to a fine powder using a pestle and mortar, and was solubilized in 1 mL double-strength SDS sample buffer (20% glycerol, 282 mm Tris base, 212 mm Tris-HCl, 4% SDS, 1.02 mm EDTA, 0.44 mm bromophenol blue and 0.35 mm phenol red) with 50 mm DTT. The sample was then heated for 10 min at 70 °C, and was centrifuged for 5 min at 10 000 g. The supernatant was collected, and 5 µL was loaded onto a NuPAGE 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) for separation by SDS–PAGE. Proteins from the gel were blotted onto either PVDF (for detection of AOX, porin and UCP) or nitrocellulose (for detection of COXII). The blots were then blocked in Tris-buffered saline plus Tween (TBST) (25 mm Tris-HCl, 125 mm NaCl and 0.1% Tween 20) with 5% skim milk powder. To probe for AOX and porin, blots were incubated overnight in antibody buffer (TBST with 5% skim milk powder) with either purified IgG against AOX isolated from a mouse hybridoma line that was originally produced by Dr Tom Elthon (University of Nebraska, Lincoln, NE, USA), at a dilution of 1:500, or purified IgG against porin isolated from a mouse hybridoma line (originally produced by Dr Tom Elthon, University of Nebraska), at a dilution of 1:5000. The blots were then washed in TBST and were incubated for 1 h in antibody buffer with alkaline phosphatase-conjugated anti-mouse antibody at a dilution of 1:3000. The blots were then washed in TBST, and the proteins were visualized using alkaline phosphatase (Promega, Madison, WI, USA); the blots were quantitated using ImageQuant (Molecular Dynamics, Sunnyvale, CA, USA). To probe for COXII, blots were incubated for 1 h in Western Blot Signal Enhancer (Vicgene, Mountain View, CA, USA), plus 5% skim milk powder, with COXII antibody (Dr Vaughan Hurry, Umeå University, Umeå, Sweden; Campbell et al. 2007) at a dilution of 1:10 000. The blots were then washed in TBST and were incubated for 1 h in Western Blot Signal Enhancer (plus 5% skim milk powder) with alkaline phosphatase-conjugated anti-rabbit antibody at a dilution of 1:3000. Proteins were detected using the AttoPhos AOP Fluorescent Substrate System (Promega); fluorescence was visualized using a fluoroimager, and the proteins were quantitated using Image J (NIH, URL: http://rsb.info.nih.gov/ij/). To probe for the UCP, the membrane was blocked in Roche's (Roche Diagnostics Corporation, Indianapolis, IN, USA) blocking reagent (Chemiluminescence Western Blotting Kit) for 1 h. The membrane was then incubated for 1 h in antibody buffer [TBS plus 0.1% (v/v) tween-2] with antibody raised against soybean UCP (Considine, Daley & Whelan 2001), at a dilution of 1:2000. The membrane was then washed in TBS and was incubated for 1 h in antibody buffer [0.1% (v/v) tween-2] with anti-rabbit antibody at a dilution of 1:10 000. The proteins were detected using the BM Chemiluminescence Western Blotting Kit, visualized using an LAS-1000 (FujiFilm, Tokyo, Japan), and quantitated using the Image Guage v4.22 software (Fujifilm).