Figure 4. Knockdown of NHERF1 and NHERF2 does not affect C3aR internalization.

(A) shRNA control HMC-1 cells (B) NHERF1 knockdown (KD) and (C) NHERF2 KD cells were exposed to buffer or C3a (100 nM) for 5 min. Cells were washed with ice-cold FACS buffer, incubated with a mouse anti-C3aR antibody or an isotype control antibody followed by PE-labeled donkey anti-mouse IgG antibody and analyzed by flow cytometry. Representative histograms plots from an internalization experiment following exposure to buffer (red line) or C3a (blue line) in shRNA control and NHERF KD cells are shown. (D) shRNA control, NHERF1 KD and NHERF2 KD cells were exposed to C3a for different time periods and receptor internalization was determined as described above. Internalization is expressed as the percentage loss of C3aR following exposure to C3a. Data represent the mean ± SEM from three experiments.