Endogenous I-2 concentrates in the primary cilium before alpha-tubulin acetylation. (A – F): ARPE-19 cells were fixed before confluence (A and B), 10 hrs after confluence (C and D), and 48 hrs after confluence (E and F), then stained for DNA (blue) + acetylated-tubulin (orange) (A, C and E) and co-stained for endogenous I-2 (green) (B, D and F). In C and D, white arrows point to the primary cilia where I-2 was present but acetylated tubulin was not detected. (G and H): ARPE-19 cells were fixed at 10 hrs after confluence, and triple stained for DNA (blue), beta-tubulin (orange) and endogenous I-2 (green). White arrow points to the primary cilia where both I-2 and beta-tubulin were present. Scale bars = 25 micron.