American Society of Hematology

Figure 5.

$Figure 5. K5 transport to the cell surface is required for removal of pre-existing CD31 from the plasma membrane. (A) CD31 surface expression was monitored by flow cytometry of AdTet- or AdK5-transduced E-DMVECs at 20 hours after infection. Where indicated, cells were treated with BFA (10 μg/mL) shortly after adenovirus infection. Uninfected E-DMVECs were stained for CD31 as control (shaded). (B) E-DMVECs were infected with AdTet, AdK5, or AdDN-PACS-2, and proteins at the cell surface were biotinylated immediately after infection and prior to K5 expression. Cells were washed 3 times in cold PBS and incubated with medium in the presence or absence of BFA. Cells were lysed after 20 hours and biotinylated proteins were captured using NeutrAvidin followed by SDS-PAGE separation, transfer to nylon membrane, and immunoblot with anti-CD31. (C) Untreated E-DMVECs (EC) or E-DMVECs transduced for 24 hours with AdK5 or AdMK3 were BFA treated or untreated and biotinylated as in panel B. Cell lysates were split in 3 fractions. Top panel shows biotinylated proteins precipitated with NeutrAvidin and immunoblotted with anti-FLAG to detect K5 or MK3. Middle panel shows precipitated, biotinylated proteins immunoblotted with antitransferrin receptor as control. Bottom panel shows total amounts of K5 and MK3 detected in cell lysates using anti-FLAG for both immunoprecipitation and immunoblotting. (D) Cells were transduced with AdTet, AdK5, AdDynK44A, or AdAP-2 as shown, and cell-surface proteins were biotinylated. Where indicated, concanamycin A or lactacystin were added to the medium during biotinylation. Biotinylated CD31 was detected by immunoblot as in panel C. (E) E-DMVECs were transduced with AdK5 or AdMK3 or untreated (EC) for 20 hours. Where indicated, the lysosomal inhibitor concanamycin A was added for 3 hours prior to immunoprecipitation of CD31 followed by immunoblotting with antiubiquitin.$

K5 transport to the cell surface is required for removal of pre-existing CD31 from the plasma membrane. (A) CD31 surface expression was monitored by flow cytometry of AdTet- or AdK5-transduced E-DMVECs at 20 hours after infection. Where indicated, cells were treated with BFA (10 μg/mL) shortly after adenovirus infection. Uninfected E-DMVECs were stained for CD31 as control (shaded). (B) E-DMVECs were infected with AdTet, AdK5, or AdDN-PACS-2, and proteins at the cell surface were biotinylated immediately after infection and prior to K5 expression. Cells were washed 3 times in cold PBS and incubated with medium in the presence or absence of BFA. Cells were lysed after 20 hours and biotinylated proteins were captured using NeutrAvidin followed by SDS-PAGE separation, transfer to nylon membrane, and immunoblot with anti-CD31. (C) Untreated E-DMVECs (EC) or E-DMVECs transduced for 24 hours with AdK5 or AdMK3 were BFA treated or untreated and biotinylated as in panel B. Cell lysates were split in 3 fractions. Top panel shows biotinylated proteins precipitated with NeutrAvidin and immunoblotted with anti-FLAG to detect K5 or MK3. Middle panel shows precipitated, biotinylated proteins immunoblotted with antitransferrin receptor as control. Bottom panel shows total amounts of K5 and MK3 detected in cell lysates using anti-FLAG for both immunoprecipitation and immunoblotting. (D) Cells were transduced with AdTet, AdK5, AdDynK44A, or AdAP-2 as shown, and cell-surface proteins were biotinylated. Where indicated, concanamycin A or lactacystin were added to the medium during biotinylation. Biotinylated CD31 was detected by immunoblot as in panel C. (E) E-DMVECs were transduced with AdK5 or AdMK3 or untreated (EC) for 20 hours. Where indicated, the lysosomal inhibitor concanamycin A was added for 3 hours prior to immunoprecipitation of CD31 followed by immunoblotting with antiubiquitin.

This Feature Is Available To Subscribers Only