Fig. 8: Ubiquitination of PFN1 regulates F-actin assembly.

a In vivo ubiquitination assay with 293T cells transfected with HA-Ub, Flag-Nedd4 and GFP-Pfn1, Pfn1-K70A, Pfn1-K108A or Pfn1-K127A. Protein lysates were immunoprecipitated with GFP-trap beads. Ubiquitinated PFN1 (Ub-Pfn1) was probed with anti-HA antibody. Uncropped images are provided as a Source data file. b Quantitation of ubiquitination levels from 5 independent experiments. Data are presented as mean +/− SEM. *P = 0.017 for polyubiquitination of Pfn1-K127A, P = 0.0009 for monoubiquitination of Pfn1-K108A and P = 0.007 for monoubiquitination of Pfn1-K127A as determined by type two Student’s t test. Source data are provided as a Source data file. c Cycloheximide (CHX) chase analysis of GFP-PFN1, PFN1-K70A and PFN1-K108A expressed in NCCs. Quantitation of protein levels after 24 h from 3 separate WT NCC lines expressing each PFN1 mutant, n = 3 independent experiments. Data are presented as mean +/− SEM. *P = 0.049 as determined by type one Student’s t test. d Representative images of F-actin (red) and stress fibre recovery after 120 min of cytochalasin D washout in control NCCs or NCCs expressing GFP-Pfn1, Pfn1-K70A and Pfn1-K108A (green). Nuclei were visualised with DAPI (blue). Scale bar = 10 μm. Quantitation of average F-actin level in NCCs after 120 min cytochalasin D washout. n = 3 independent experiments. Data are presented as mean +/− SEM. **P = 0.007 as determined by type one Student’s t test.