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Fig. 2 - Supplemental 4 Optimized Halo-PafA and Pup substrates efficiently promote pupylation in vitro in a proximity-dependent manner. (A) Comparison of proximity-tagging by different Halo-PafA derivatives on in vitro pupylation using TSK8R-sPupK61R. (B) Comparison of different Pup substrates, SBPK4R-sPupK61R and TSK8R-sPupK61R, on in vitro pupylation. All reactions were conducted with 1 μM of a Halo-PafA derivative, 10 μM of a sPup substrate, and 0.5 μM of a purified short SRC with a 2xV5 tag, SRC(247-536)-2xV5, at 37°C for 30 min. Pupylation levels of SRC were assessed by immunoblot (IB) using an anti-V5 antibody.