Fig 1: Activation of the Complement Cascade in Presence of Ad5 and 9C12 Results in Deposition of C4b on the Ad5 Capsid(A) Western blot of C4 (a-chain) cleavage in NHS (top) or C1q-depleted serum (bottom) in the presence of Ad5 and 9C12-WT over 60 min. See also Figure S2E.(B) Experimental set up (left): Ad5-mCherry+9C12-WT were incubated with HBS++/serum for 1 h at 37°C. Ad5-GFP was incubated with HBS++. Ad5-mCherry was added to HeLa TRIM21 KO cells immediately followed by Ad5-GFP. Right: relative infection of Ad5-mCherry and Ad5-GFP. x axis labeling corresponds to the buffer/serum that was incubated with Ad5-mCherry.(C) Western blot of C4 (a-chain) cleavage in NHS, C1q-depleted serum, or C4-depleted serum in the presence of Ad5 and 9C12-WT as indicated.(D) Elisa for C4 using serum (left) or purified protein (right). Ad5+9C12-WT were incubated with the indicated serum or purified protein and pelleted over a sucrose gradient.Error bars depict mean + SD of three replicates acquired in one representative experiment (A and D). Original western blots are included in Figure S6.