Fig. 1: Isolation protocol and characterization of the APF from Arabidopsis leaves. | Nature Communications

Fig. 1: Isolation protocol and characterization of the APF from Arabidopsis leaves.

From: Proteasomes accumulate in the plant apoplast where they participate in microbe-associated molecular pattern (MAMP)-triggered pathogen defense

Fig. 1

a Protocol for isolating the leaf APF. Non-damaged 5-6-week-old leaves were vacuum infiltrated with extraction buffer containing ATP under a mild negative pressure with the infiltrate then collected by low g centrifugation of vertically hung leaves. Clarification of the fluid using a 0.2-μm cut-off filter generated the APF, whose vesicles and large particles were then concentrated if needed by centrifugation at 100,000 Xg, resulting in the APF pellet (APFp). Created in BioRender. Karimi, H. (2025) https://BioRender.com/r81f161. b Level of chloroplast contamination in the APF as compared to the crude leaf lysate (CL) by spectrophotometric assays for chlorophyll. Bars reflect the mean (±SD) of three technical replicates. Individual data points are included. c The distribution of protein, cytosolic FBP, actin, and the Rubisco large subunit in the CL and APF by staining for protein with silver or by immunoblot analysis with specific antibodies. MW, molecular mass markers.

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