Fig. 3.

Effect of calpain inhibition on PA internalization. (A and B) RAW264.7 cells were incubated with 80 µM MDL28170 or DMSO control for 1 h. The cells then were exposed to 0.5 µg/mL PA and maintained at 4 °C for 1 h for binding assays (A) or at 37 °C for the indicated time for internalization assays (B). Cell lysates were analyzed by Western blotting using anti-PA antibody as described in Materials and Methods. The SDS-resistant PA oligomer signal was quantified by normalization against actin, and the resulting value was compared with the signal obtained from DMSO-treated cells at time point 0.5 h (which was assigned a value of 1.0). The mean ± SD for values obtained in three independent experiments are shown below the lanes in the Western blot in B. (C) Immunofluorescence analysis of cellular localization of PA and β₁ integrin during PA internalization. RAW264.7 cells were preincubated with or without 80 µM MDL28170 for 1 h, followed by exposure to Alexa-Fluor 488–labeled PA in the presence of 0.5 µg/mL anti–β₁ integrin-APC (HMβ1–1) antibody at 37 °C for 20 min. (Left) The cellular localization of PA (green fluorescence) and β₁ integrin (red fluorescence) was monitored microscopically. (Right) The intensities of cytoplasmic PA and β₁-integrin signal were quantified by ImageJ analysis as described in Materials and Methods. Data represent mean ± SEM values (n > 30). ***P < 0.001.