Journal: Journal of Virology
Article Title: Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt
doi: 10.1128/JVI.00167-20
Figure Lengend Snippet: HCMV induces a chronic SHIP1 activation required for the survival of infected monocytes. (A) Monocytes were mock or HCMV infected for 15 min, 24 h, or 48 h, and total or p-SHIP1 was detected from whole-cell lysates. (B, C, and D) Monocytes were treated with 5 μM AG (an EGFR inhibitor), 50 μM LY (a PI3K inhibitor), or 5 μM 3AC (a SHIP1 inhibitor) for 1 h and then mock or HCMV infected. (B) After 5 min infection, anti-gH antibody was used to pull down gH glycoprotein (and any other proteins bound to gH) followed by blotting for gH, integrin β1, SHIP1, and p-SHIP1 in the immunoprecipitate using specific antibodies. Input controls were blotted for actin to confirm homogeneous loading of the samples. (C) After 24 h, Western blot analysis was performed to detect Mcl1 and HSP27 from total cell lysates. (D) Survival of monocytes was measured after 48 h of infection with or without the inhibitors by annexin V and PI staining using flow cytometry. Results are representative of at least 3 to 5 independent experiments using different donors. **, P < 0.005.
Article Snippet: For the inhibitor studies, the following reagents were purchased from the indicated companies: AG-1478 (AG) (an EGFR inhibitor [ 96 ]), 3-α-aminocholestane (3AC) (a SHIP1 inhibitor [ 66 ]), and LY294002 (a pan-PI3K inhibitor [ 97 ]) from Calbiochem (Billerica, MA) and ATN161 (ATN) (an integrin α5β1 inhibitor [ 98 ]) from Selleck Chemicals (Houston, TX).
Techniques: Activation Assay, Infection, Western Blot, Staining, Flow Cytometry