Fig. 6: PBMs in OTU11 and OTU12 are required for their interaction with anionic lipids in vitro. | Nature Communications

Fig. 6: PBMs in OTU11 and OTU12 are required for their interaction with anionic lipids in vitro.

From: Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking

Fig. 6: PBMs in OTU11 and OTU12 are required for their interaction with anionic lipids in vitro.The alt text for this image may have been generated using AI.

a Lipid species spotted on the membrane for the lipid overlay assay. The solid dark gray and light gray spots indicate lipids that showed interactions in the lipid-overlay assays. b–d Lipid overlay assays with GST, GST-OTU11(WT), GST-OTU11(6A1), GST-OTU11(6A2), and GST-OTU11(6A1 + 6A2) (b), GST, GST-OTU12(WT), GST-OTU12(6A1), and GST-OTU12(6A2) (c) and GST, GST-OTU11(OTU), GST-OTU11(OTU-6A1), and GST-OTU11(N) (d). Bound proteins were detected with an anti-GST antibody. e Representative gel images of liposome sedimentation assays using GST-OTU11(WT) and GST-OTU12(WT). GST-fusion proteins were incubated with the liposome buffer alone or with liposomes (PC/PE) containing no PIPs or 5% of one of PI(3)P, PI(4)P, or PI(4,5)P2. SDS-PAGE gels were stained with TCE. M molecular mass marker. f Quantification of the result in (e). The signal intensity of the protein band in the pellet fraction was divided by the signal intensity of the protein band in the supernatant fraction for each lane to calculate the intensity ratio of pellet/supernatant (P/S). Box plot shows the results of the quantification of at least three independent experiments. Center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range; points, outliers, n = 3 experiments. g Representative gel images of liposome sedimentation assays of GST-OTU11(WT) and GST-OTU11(6A1). GST fusion proteins were incubated with the liposome buffer alone or with liposomes (PC/PE) containing 5% of PI(3)P, PI(4)P, or PI(4,5)P2. M molecular mass marker. h Quantification of the result in (g). Box plot shows the results of the quantification of three independent experiments. Center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range; points, outliers, n = 3 experiments. i A representative gel image of liposome sedimentation assays of GST-OTU11(WT) or GST-OTU11(N). GST-fusion proteins were incubated with liposomes (PC/PE) containing 5% of PI(4,5)P2 or with the liposome buffer alone. M molecular mass marker. j Quantification of the result in (i). Box plot shows the results of the quantification of three independent experiments. Center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range; points, outliers, n = 3 experiments. Source data are provided as a Source Data file.

Back to article page