Cadherin expression in RP1-mutants.

4A N-cadherin levels of HEK293 cells containing empty vector (c), wt, ALA and ASP were determined by immunoblotting with an N-terminal N-cadherin antibody. The middle panel shows a processed N-cadherin fragment (named CTF1) detected by a fragment specific antibody in respective lysates. The lower panel shows the β-tubulin loading control. 4B The Western blot signals of 4A were quantified using the Image-J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997–2008). Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001) comparing control versus wt and mutants regarding complete N-cadherin and comparing wt versus mutants regarding N-cadherin cleavage fragment (CTF1). In the right panel, the empty c lane indicates no detectable CTF in control cells. 4C N-cadherin levels were measured by incubation with a monoclonal antibody directed against the cytoplasmic tail and subsequent FACS analysis. The negative control (yellow line) was incubated with secondary antibody only. The positive control (red) was empty vector containing HEK293 cells. The results for HEK293 expressing RP1-wt are depicted in black, RP1-ALA²³⁶ in green and RP1-ASP²³⁶ in blue. 4D Quantification of N-cadherin levels from FACS analysis. Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001).