FIG 2.

RSV NS2 protein interacts with Beclin1 protein. (A) Coimmunoprecipitation analysis of 293HEK cells coexpressing Flag-tagged Beclin1 (Flag-Bec1) and either HA-tagged NS2 (HA-NS2), HA-tagged NS1 (HA-NS1), or empty HA vector control (HA). Beclin1 was immunoprecipitated (IP) with anti-Flag antibody, followed by immunoblotting (IB) with either anti-HA or anti-Flag antibody. Whole-cell extracts (WCE) were immunoblotted with anti-Flag, anti-HA, and anti-actin antibodies. A blue arrowhead indicates NS2 protein. (B) Coimmunoprecipitation analysis of A549 cells expressing HA-NS2. Endogenous Beclin1 was IP with anti-Beclin1 antibody, followed by IB with either anti-HA or anti-Beclin1 antibody. WCE were immunoblotted with anti-HA and anti-actin antibodies. Red arrowheads indicate Beclin1 protein. (C) Coimmunoprecipitation analysis of A549 cells expressing HA-NS2. HA-NS2 was IP with anti-HA antibody, followed by IB with either anti-HA or anti-Beclin1 antibody. WCE were immunoblotted with anti-HA and anti-actin antibodies. A red arrowhead indicates Beclin1 protein. (D) Coimmunofluorescence analysis of Beclin1 and NS2 interaction in A549 cells coexpressing Flag-Beclin1 and HA-NS2. Fixed cells were probed with either anti-Flag or anti-HA primary antibodies and subsequently labeled with fluorescent secondary antibodies: FITC (green) for Flag-Beclin1 and Alexa Fluor 594 (red) for HA-NS2. The yellow color in the merge panels indicates colocalization of NS2 with Beclin1. Immunoblot images are representative of three independent experiments. Coimmunofluorescence images are representative of 10 different fields.