Fig. 2.

Cell based assays. (A) PDZ domain–dependent colocalization of HTRA1 with CAPN2 in HEK293 cells. Doxycycline (DOX)-inducible HEK293 cells expressing HTRA1 or HTRA1ΔPDZ, respectively, were treated with DOX and transiently transfected with CAPN2. Fixed cells were immunostained against the Strep tag of CAPN2 (red) and the Myc tag of HTRA1 (green) (Scale bar: 10 µm). Colocalization was quantified using the Pearson correlation coefficient. Data represent SEM of 15 images taken from 3 independent experiments per condition. ****P < 0.0001 (Mann–Whitney U test). (B) Coimmunoprecipitation of HTRA1 with CAPN2. Lysates (Input) of cells described in (A) were used for immunoprecipitation (IP) with Strep- or Myc-conjugated beads and immunoblotted using anti-HTRA1 and anti-CAPN2 antibodies. Representative data are shown (n = 3). (C) SW480 cells stably overexpressing HTRA1 (HTRA1) or not (EV) were transiently transfected with a CAPN2 plasmid for 24 h. Cell lysates were immunoblotted using anti-ANXA1, anti-CAPN2, and anti-actin antibodies. Protein levels were quantified by ImageJ, and the ANXA1 levels were normalized by actin. Data represent mean ± SEM of 6 experiments (*P = 0.01).