NSP4 was detected in the media of intact, RV-infected MDCK and HT29.F8 cells. Cells were infected with RV SA11.4F at a MOI of 2 for 6, 8 and 12 hpi. At each time point, the media was collected, clarified to remove cell debris, and ultra-centrifuged to remove RV particles prior to separation by SDS-PAGE and analyses by Western blot. Panel A: membrane was probed with anti-NSP4150-175. The clarified media collected at 6 hpi from MDCK (lane 1) or HT29.F8 (lane 4) cells were concentrated 10-fold prior to analyses. The clarified media collected at 8 and 12 hpi from RV-infected MDCK cells (lanes 2 and 3, respectively) and HT29.F8 cells (lanes 5 and 6, respectively) were analyzed in the absence of concentration. Panel B: the same membrane was probed with anti-GAPDH as a membrane integrity control. Lane 7 is HT29.F8 cell lysates showing the presence of GAPDH (positive antibody control).