Figure 5 | Scientific Reports

Figure 5

From: Disease-associated synaptic scaffold protein CNK2 modulates PSD size and influences localisation of the regulatory kinase TNIK

Figure 5

TNIK localisation is regulated by CNK2. (A) Scheme of CNK2 truncation variants (for detailed description of domains see Fig. 4A legend). (B) Coimmunoprecipitation experiments of EGFP-CNK2 variants overexpressed in CHL V79 cells together with FLAG-TNIK. Proteins were immunoprecipitated with either anti-GFP (mouse) antibody or mouse IgGs as a negative control. Pull-down control and coprecipitated proteins were analysed by western blot with anti-GFP (IP) and anti-FLAG (coIP) antibodies. Input control (lysate) is shown on the left. (C) TNIK localisation is influenced by CNK2 in heterologous cells. Representative immunofluorescence experiments in CHL V79 cells overexpressing FLAG-TNIK together with EGFP as control (upper panel), EGFP-CNK2 (middle panel) or EGFP-CNK2ΔPH (lower panel). Left panels show EGFP only or EGFP-tagged CNK2 variants (cyan), middle panels show FLAG-TNIK (magenta), and the right lane shows merged channels. Scale bar: 20 µm. (D) For quantification of TNIK localisation in CHL V79 cells reflecting the experiments shown in (C), images expressing both proteins, were randomised and classified according to cytosolic or membranous TNIK localisation. Co-expression of CNK2 recruits the main fraction of FLAG-TNIK to the membrane. Upon co-expression of EGFP-tagged CNK2 variants lacking the membrane binding region, FLAG-TNIK shows essentially no membranous localisation. Data used for quantification include data for a total of 73–114 cells per condition, from three independent experiments.

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