MC4 | Metacaspase-4

10 day old Arabidopsis thaliana seedlings, Ler wild type (WT), Col-0 (WT) and MC4 T-DNA insertion mutants Ler mc4 and Col-0 mc4. Grown on 1⁄2 MS medium (no addition of sugar). • Complete seedlings were grinded on liquid nitrogen and tissue powder was immediately mixed with denaturing Laemmli buffer preheated at 70 °C and further denatured at 70 °C for 10 min (= 0 minutes of wounding). Alternatively, tissue powder was kept at room temperature for 5 minutes and mixed with denaturing Laemmli buffer preheated at 70 °C and further denatured at 70 °C for 10 min (= 5 minutes of wounding). • An equal volume of leaf grinded on liquid nitrogen was mixed with denaturing 2x Laemmli buffer preheated at 70 °C and further denatured at 70 °C for 10 min. Samples were separated on 12 % SDS-PAGE (stainfree, BioRad) and blotted 7 minutes to PVDF (pore size of 20 um), using semi-dry (BioRad Turboblot) . Blot was blocked with 5% milk for 2h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 ON/4°C in PBS-T, 1% milk, with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed three times for 10 min in PBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (Goat anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:10 000 in PBS-T, 1% milk, for 2h/RT with agitation. The blot was washed as above and developed for 1 min with Agrisera ECL Superbright. Exposure time was 1-10 seconds.

Clear band at correct height of approximately 55 kDa in Col-0 at 0 minutes of wounding. After 5 minutes, MC4 autocatalytically cleaves itself to be activated, so the full length MC4 band at 55 kDa disappears.
This band is clearly absent in Col-0 mc4 which is a clear evidence for specific detection of MC4 in Col-0.

Courtesy Dr. Simon Stael,VIB-Ugent Center for Plant Systems Biology, Ghent University, Belgium