Fig. 3: PUX3/4/5 selectively associate with the INM via interaction with the nucleoskeleton.

a Transformed and averaged PSM data of all AtPUX proteins from PL-LFQMS profiling using Arabidopsis plants expressing BioID2-tagged INM protein SUN1, NEAP1, and NEMP_A. Three biological replicates were used. Asterisks (**, ***) represent significant enrichment with p-value < 0.01 and 0.001 (linear model F-test) compared to controls. b SUN1 specifically associates with PUX4 and PUX5 but not PUX7. GFP-SUN1 was transiently coexpressed with PUX4-HA, PUX5-HA, or PUX7-HA in N. benthamiana. Protein extract was immunoprecipitated with anti-HA antibody-conjugated agarose beads before immunoblotting with anti-HA and anti-GFP antibodies. c PUX4 associates with SUN1 but not with ARA6, WIT1, and BRI1. PUX4-HA was transiently coexpressed with GFP-SUN1, GFP-WIT1, ARA6-GFP, or BRI1-GFP in N. benthamiana. Protein extract was immunoprecipitated with anti-HA antibody-conjugated agarose beads before immunoblotting with anti-HA and anti-GFP antibodies. d Scatter plot showing significantly enriched proteins (red dots) identified by PL-LFQMS profiling using PUX5 as the bait. PUX5-BioID2-HA without biotin treatment (PUX5−) and YFP-BioID2-HA without biotin treatment (YFP−) were used as controls. Three biological replicates were used. Candidates with PSM > 2 were further filtered using cutoffs log₂FC > 1.5 and p-value < 0.01 (linear model F-test) compared to both controls. e BiFC assays showing interactions between PUX3/4/5 and KAKU4 at the nuclear periphery. BiFC YFP constructs together with free mCherry were transiently coexpressed in N. benthamiana, and epidermal cells were imaged. Bars = 10 μm.