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. Author manuscript; available in PMC: 2021 Apr 8.
Published in final edited form as: Cell Host Microbe. 2020 Feb 25;27(4):629–641.e4. doi: 10.1016/j.chom.2020.01.024

Figure 2. Qtip prevents in vitro cleavage of cIVP882 but cIVP882 mutants lacking cleavage or catalytic sites do not prevent Qitp recognition.

Figure 2.

(A) In vitro cleavage of cIVP882-HALO monitored by SDS-PAGE analysis. The presence of RecA and/or ATP-γ-S in the reactions is indicated above each lane. (B) In vitro cleavage of cIVP882-HALO purified alone or in complex with HIS-Qtip. Samples were concentration matched according to the cIVP882-HALO Alexa660 signal. (C) In vitro cleavage of WT cIVP882-HALO and the indicated catalytic-site (K172A and S130A) and cleavage-site (G92D) variants. For (A-C): all cIVP882-HALO proteins were labeled with HALO-Alexa660. Upper panels, gels imaged using a Cy5 filter set for HALO-Alexa660 detection; lower panels, the same gels stained for total protein. Incubation times noted above each lane. Marker, M. In (B) and (C), all samples contained ATP-γ-S and RecA. (D) Composite images from individual cell analyses of E. coli producing Qtip and either WT cIVP882-HALO or the cleavage-site (G92D) or catalytic-site (S130A and K172A) cIVP882-HALO variant. HALO-TMR fluorescence intensity and scale bar represented as in Figure 1D. See also Figure S1.