Fig. 4: NEDD4 ubiquitinates PFN1. | Nature Communications

Fig. 4: NEDD4 ubiquitinates PFN1.

From: Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells

Fig. 4

a In vivo ubiquitination assay with 293T cells transfected with HA-Ub, GFP-Pfn1 and Flag-Nedd4 or Flag-Nedd4-CS. Protein lysates were immunoprecipitated (IP) with GFP-trap beads. Ubiquitinated PFN1 (Ub-Pfn1) was probed with anti-HA antibody. Quantitation of the ubiquitinated species was performed from 4 independent experiments. Arrow indicates monoubuiqitinanted PFN1. Data are presented as mean +/− SEM. *P = 0.035 as determined by type two Student’s t test. Uncropped images are provided as a Source data file. b 293T cells were transfected with various combinations of Flag-Nedd4, Flag-Nedd4-CS and GFP-Pfn1. NEDD4 and NEDD4-CS were precipitated from protein lysates with anti-Flag antibodies and compared to mouse IgG. Corresponding antibodies were used to recognize co-immunoprecipitated proteins. Uncropped images are provided as a Source data file. Blots are representative of 3 repeats. c Single-guide RNA (sgRNA) sequence designed to target exon 2 of Nedd4 for gene knockout by CRISPR. Western blot of NCU10K NCC clones derived from single cells confirms complete KO of NEDD4 and altered abundance of PFN1. Quantitation of PFN1 levels from n = 3 biologically independent replicates. Data are presented as mean +/− SEM. *P = 0.038 as determined by type-one Student’s t test. Uncropped images are provided as a Source data file. d In vivo ubiquitination assay with WT and Nedd4−/− NCCs transfected with GFP-Pfn1. Protein lysates were immunoprecipitated with GFP-trap beads and ubiquitination detected with anti-Ub antibodies. Quantitation of ubiquitin levels on immunoprecipitated GFP-PFN1 from 3 WT and 3 Nedd4−/− NCC lines. Data are presented as mean +/− SEM. *P = 0.048 as determined by type-two Student’s t test. Source data are provided as a Source data file. e Cycloheximide (CHX) chase analysis of Pfn1 degradation in WT and Nedd4−/− NCCs. Blots are representative of 3 separate experiments. CQ, chloroquine. f Quantitation of cycloheximide chase assay from n = 3 biologically independent replicates. Data are presented as mean +/− SEM. *P = 0.043 as determined by type one Student’s t test. Source data are provided as a Source data file.

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