Perfect blue web m

Thylakoid proteins were separated by SDS-PAGE and then transferred to a polyvinylidene membrane (Hybond P, GE Healthcare) using a tank blot system (Perfect Blue Web M, VWR International GmbH, Darmstadt, Germany). Immunochemical detection was performed using an enhanced chemiluminescence detection reagent (ECL Prime, GE Healthcare) according to the manufacturer’s instructions. Chemiluminescence was detected using the G:BOX XT4 imaging system (Syngene, Cambridge, UK). The following specific antibodies against the photosynthetic proteins were purchased from Agrisera AB (Vennäs, Sveden): PsbB (order number: AS04 038), PsbD (AS 06 146), LHCB1, LHCB4 (AS04 045), PetA (AS06 119), PetB (AS03 034), PETC (AS08 330), PsaA (AS06 172), PsaB (AS10 695), PSAD (AS09 461), PSAF (AS06 104), PSAH (AS06 105), PSAK (AS04 049), PSAL (AS06 108), PSAN (AS06 109), LHCA1 (AS01 005), LHCA2 (AS01 006), LHCA3 (AS01 007), LHCA4 (AS01 008), and AtpB (AS05 085). Antibodies against Ycf4 and Y3IP1 were produced by Krech et al. (2012) . To determine protein oxidation levels, the Millipore Oxyblot^TM protein oxidation detection kit (Merck Chemicals GmbH, Darmstadt, Germany) was used according to the manufacturer’s protocol. Thylakoid proteins equivalent to 2 µg chlorophyll were loaded per reaction.

Thylakoid proteins were separated by SDS-PAGE and then transferred to a polyvinylidene membrane (Hybond P, GE Healthcare) using a tank blot system (Perfect Blue Web M, VWR International GmbH, Darmstadt, Germany). Immunochemical detection was performed using an enhanced chemiluminescence detection reagent (ECL Prime, GE Healthcare) according to the manufacturer’s instructions. Chemiluminescence was detected on X-ray film. Antibodies against the photosynthetic proteins were purchased from Agrisera AB (Vännäs, Sweden).

Mitochondrial proteins were extracted according to published protocols [65 (link)] with a few modifications. The extraction buffer was supplemented with 0.02% N-lauroylsarcosine and 0.02% Triton X-100 [66 (link)]. 0.75, 1.5, 3 and 6 µg of mitochondrial proteins were separated by electrophoresis on 15% SDS-containing polyacrylamide gels. The gels were either stained with Coomassie Brilliant Blue (Serva, Heidelberg, Germany) or blotted onto polyvinylidene difluoride membranes (Hybond-P, GE Healthcare, Buckinghamshire, UK). For blotting experiments, the separated proteins were transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK) using the tank blot system (Perfect Blue Web M, PeqLab, Erlangen, Germany) and a standard transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3). Membranes were treated with blocking buffer (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20, and 0.5% BSA) overnight and subsequently incubated for 1 h with COX2 monoclonal antibody, diluted in buffer (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20). COX2 antibody dilution was 1:1000. Secondary rabbit anti-mouse IgG conjugated to horseradish peroxidase were detected with the ECL Plus proteins gel blotting detection system (GE Healthcare, Buckinghamshire, UK).