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. 2015 Aug 26;27(4):1159–1173. doi: 10.1681/ASN.2014111074

Figure 6.

Figure 6.

ARRB1/2 and AP2 mediate cAMP-mediated endocytosis of PC1. (A and B) Live–cell surface labeling of full-length PC1 in HEK293 cells cotransfected with scrambled-, AP2M1-, or ARRB1 1/2–specific siRNA. A significant inhibition of cAMP–stimulated PC1 endocytosis was observed with AP2M1 or ARRB1 siRNA–treated cells compared with scrambled siRNA–transfected cells. ***P<0.001. Scale bar, 10 µm. (C and D) Live–cell surface labeling of full–length PC1-S3164D in HEK293 cells cotransfected with scrambled-, AP2M1-, or ARRB1 1/2–specific siRNA. PC1-S3164D surface expression was significantly increased in cells transfected with AP2M1 or ARRB1/2 siRNA compared with scrambled siRNA–transfected cells. ****P<0.0001. Scale bar, 10 µm. (E and F) Surface biotinylation of PC1-S3164D in HEK293 cells showed that siRNA knockdown of AP2M1 or ARRB1/2 resulted in an increase in surface-labeled PC1 compared with scrambled siRNA controls. Arrowheads indicate full-length (FL) or cleaved C–terminal fragment (CTF) as detected by an HA blot. Controls included the PM transporter Na+-K+-ATPase and the ER resident protein calnexin. The bars show the ratio of biotinylated PC1 to total PC1 (CTF; HA blot) from three experiments. **P<0.01; ***P<0.001.