Media fractions were collected and centrifuged at 1000×g for 0h 10m 0s

The supernatant fractions were collected and concentrated (20-fold) using a 10 kDa Amicon filter.

Concentrated media fractions (1 ml) were incubated with 20 μl of anti-FLAG M2 affinity gel for 1h 0m 0s at 4°C

After washing 5 times with lysis buffer , SDS-PAGE sample loading buffer was added to the beads (twice the beads volume) and samples were processed for SDS-PAGE and immunoblot.