HAI-1 and HAI-2 both promote matriptase plasma membrane translocation despite their different subcellular distributions. Teton HAI-1 cells, Teton HAI-2 cells, and the TA control were grown in the absence (Dox -) or presence (Dox +) of 1ug/ml doxycycline for 24 h. These cells were then subjected to biotinylation of cell surface proteins, followed by lysis of the cells and the depletion of biotinylated cell surface proteins from the lysates using avidin-agarose beads. The lysates prior to (lanes 1 and 3, T) and after (lane 2 and 4, D) depletion of biotinylated cell surface proteins were analyzed by immunoblot for matriptase (A), HAI-1 (C, left panel), HAI-2 (C, right panel), and GAPDH as a loading control. The ratios of cell surface matriptase, HAI-1, and HAI-2 relative to their respective total expression levels are presented in panels (B) and (D). These data are representative of more than 3 independent experiments. ns: non-significant difference; ∗: P-value of comparison without Dox treatment; ∗∗∗: P < 0.001; #: P-value of comparison with Dox treatment; #: P-value < 0.001. The protein bands of HAI-1 and HAI-2 were indicated.