Fig. 1: CaLB1 interacts with ALIX. | Nature Communications

Fig. 1: CaLB1 interacts with ALIX.

From: Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation

Fig. 1

a alix mutants accumulate autophagosome markers. Total extracts of 7-day-old wild type, alix-4, alix-2 and atg7-2 were subjected to immunoblotting using an anti-ATG8 and anti-NBR1 antibodies. An anti-CDC2-antibody was used as a loading control. The experiment was conducted twice and one of the results is shown. Constructs of CaLB1 (b) and Arabidopsis ALIX (c) used for the interaction studies. FL: full length; C2: C2 ___domain; P/PRD: proline-rich ___domain; 1-177: CaLB1 fragment 1-177; 1-220: CaLB1 fragment 1-220; ∆C: ALIX lacking the C-terminus; BRO1: Bro1-___domain; ∆BRO1: ALIX lacking the Bro1-___domain. d, e Yeast two-hybrid analysis between GAD-fused CaLB1 and GBD-fused ALIX. Results of three independent transformants are shown. -LW: media without leucine and tryptophan, -LWH: media without leucine, tryptophan, and histidine, 3-AT: 3-Amino-1,2,4-triazole. The experiment was conducted three times, and one representative result is shown. f in vitro binding assay between CaLB1-6xHis and MBP-ALIX. MBP-fused ALIX (BRO1) and ALIX(∆BRO1) were mixed with CaLB1-6xHis bound to TALON beads. After incubation beads were washed and bound proteins were eluted and subjected to immunoblots using an anti-MBP antibody and an anti-His antibody. The experiment was conducted three times, and one representative result is shown. g in vitro binding assay between CaLB1(C2)−6xHis or CaLB1 (FL)−6xHis and GST-ALIX. His-tag fused CaLB1 proteins were mixed with GST-ALIX bound to glutathione Sepharose. After incubation and extensive washing, bead-retained proteins were eluted and subjected to immunoblots using an anti-GST antibody and an anti-His antibody. The experiment was conducted twice, and one representative result is shown. h Root epidermal cells of seedlings expressing both ALIXpro: GFP-ALIX and CaLB1pro: CaLB1-mRFP were analyzed under a confocal microscope. 485 CaLB1-mRFP foci from 17 images were analyzed and 35.3% of the mRFP-positive showed colocalization with GFP-ALIX. Scale bars: 10 µm. The magnified region is indicated with a white rectangle. Scale bar in the magnification: 1 µm. a, f, g Source data are provided as a Source Data file.

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