Figure 7: Analysis of N-WASP as a downstream effector.
From: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology
(a) GST-Pak1-PBD used to pull down GTP-Cdc42 from fibroblast lysates (upper panel). Lower panel, total Cdc42 levels. Mouse monoclonal Cdc42 antibody detects Cdc42. GAPDH used for normalization. *indicates longer exposure for the pull down and note that the wells loaded with pull down samples from WT and KO were separated by one well in between for visualizing distinct bands. (b) Densitometric analysis of active Cdc42 levels. The bar chart shows fold decrease in activated levels as a ratio of the active to the total levels (n = 4 independent experiments, mean ± SD; *P < 0.05). (c) GFP microbeads used to precipitate GFP-CRN7 from HEK293T cell extracts, the precipitate probed with rabbit polyclonal anti-N-WASP antibodies. GFP used as control. (d) Immunofluorescence analysis of HEK293T cells expressing GFP-CRN7 (green). Staining with N-WASP pAb (red) for endogenous protein. Yellow, merge, co-localisation. Scale bar = 25 μm. (e) Schematic showing GST fusion polypeptides of CRN7 (NT and CT). The positions of the amino acids are indicated. (f) Affinity-purified GFP-tagged full-length N-WASP blotted to nitrocellulose membranes directly bound by GST-NT-CRN7 and CT-CRN7 but not by GST (left-most panel). *indicates longer exposure of that particular strip of membrane. N-WASP additionally visualized by anti-GFP immunoblotting (whole right panel). (g) GFP-CRN7 and its NT and CT fragments precipitated from HEK293T cell extracts using GFP microbeads, N-WASP detected with pAb N-WASP. mAb K3-184-2 verified immunoprecipitated proteins. GFP used as negative control in (c,f,g).
