Proteomic profiling of GFP-tagged PIN-LIKES (PILS2, PILS3, PILS6) in Arabidopsis root extracts reveals ERAD components as major interactors

This dataset describes a proteomic analysis of proteins co-immunoprecipitated with functional GFP-tagged PILS2, PILS3, and PILS6 fusion proteins in Arabidopsis thaliana roots. PILS (PIN-LIKES) proteins are auxin transport facilitators that localize to the endoplasmic reticulum (ER). To identify proteins that associate with PILS2, PILS3, and PILS6 under physiological conditions, we performed GFP-based immunoprecipitation (IP) followed by mass spectrometry (MS). Protein extracts were prepared from root tissues of Arabidopsis Col-0 (wild-type) and PILS2-, PILS3-, and PILS6-overexpressing transgenic lines expressing N- or C-terminal GFP fusion proteins. Plant material was ground in liquid nitrogen, and total proteins were extracted using aTris Buffer containing 1% CHAPS, 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 15 mM EGTA, 75 mM NaCl, 1 mM DTT, 0.1% Tween-20, and protease inhibitors. Extracts were centrifuged, and the supernatants were quantified using the Bradford assay. 2mg of protein were subjected to immunoprecipitation using anti-GFP magnetic beads, Col-0 (non-tagged) extracts were used as negative controls. Co-immunoprecipitated proteins were then processed and analyzed by mass spectrometry.