Figure 7
The CaNDR1a protein is enriched in plasma membrane fraction. The HA-CaNDR1a construct (see Figure 5a) was used for carrying out two independent experiments that consisted of two independent agroinfiltrations and plasma membrane (PM) preparations. A representative experiment is presented in this figure. Agroinfiltration and immunoblot conditions are described in the 'Methods' section. (a) Detection of HA-CaNDR1a proteins in Agrobacterium-infiltrated leaf tissues. Crude extract (CE) was prepared by directly incubating tissues at 95°C for 5 min in 1X Laemmli buffer [57]. (b) Detection of HA-CaNDR1a proteins and endogenous PM-resident proteins PMA2 in microsomal and PM fractions. PMA2 is a proton-ATPase pump previously shown to be localized exclusively at the PM [61]. Membrane was probed using a specific anti-PMA2 serum [58] in order to check for the purity of the PM fraction. As expected, PMA2 proteins appeared to be significantly enriched in the PM fraction versus the microsomal (μ) one, as also observed for HA-CaNDR1a proteins upon stripping and reprobing of the same blotting membrane with HA-specific antiserum (Middle panel). Membranes were also stained with Ponceau S to show the equal loading between both fractions, i.e. μ and PM (lower panel). (c) HA-tagged CaNDR1a proteins are not detected in soluble fractions. Distinct protein amounts of soluble (100.000 × g supernatant) and PM fractions (5, 10 and 15 μg) were resolved by SDS-PAGE and immunoblotted using a HA-specific antiserum.