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. Author manuscript; available in PMC: 2021 Mar 13.
Published in final edited form as: Circ Res. 2020 Feb 13;126(6):737–749. doi: 10.1161/CIRCRESAHA.119.315760

Figure 2. Validation of the cut and paste method for rapid removal and replacement of cMyBP-C N’-terminal domains.

Figure 2.

A) Western blot of LV homogenates probed with an antibody to cMyBP-C before and after TEVp treatment and after covalent bond formation with rC0C7-sc. Uncut cMyBP-C is visible as a band (green) in the untreated lane (left). After TEVp treatment the gC0C7 proteolytic fragment (yellow box) can be removed by washing with fresh solutions. Newly ligated rC0C7-sc-st-C8C10 protein is visible as a band (green) in lanes with added rC0C7-sc. Excess (un-ligated) rC0C7-sc is visible as a lower molecular weight band (green) below cMyBP-C. α-actinin (red) served as a loading control. B) Control experiment showing a western blot of cMyBP-C in WT myocytes before and after TEVp treatment and after addition of rC0C7-sc. WT cMyBP-C was not cleaved by TEVp and addition of rC0C7-sc did not affect the native (WT) cMyBP-C band. C) Immunofluorescence staining showing the normal doublet (green) pattern of cMyBP-C staining in HO myocytes (left panel), loss of green doublets after TEVp treatment (middle panel), and reappearance of doublets after ligation with rC0C7-sc (right panel). Control experiments showed TEVp had no effect on the doublet pattern of cMyBP-C localization in WT myocytes (Inset, middle panel) and addition of rC0C7 (without SpyCatcher) did not restore cMyBP-C doublets (Inset, right panel). D) Representative western blots showing ligation efficiency of rC0C7-sc in TEVp treated LV homogenates from HO Spy-C mice. Top, Homogenates were probed with an antibody against cMyBP-C. Middle and bottom panels, western blots of LV homogenates probed with a custom antibody against SpyTag. The SpyTag antibody recognized SpyTag in cMyBP-C prior to TEVp treatment (middle panel, green band in left untreated lane) and also recognized SpyTag in the smaller st-C8C10 (~35 kDa) fragment after TEVp treatment (bottom panel). However, the SpyTag antibody did not recognize SpyTag in the ligated rC0C7-sc-st-C8C10 protein after covalent bond formation with SpyCatcher (note the absence of green cMyBP-C bands in the middle panel in all TEVp treated + rC0C7-sc lanes). E) Summary data from western blots as in D to quantify efficiency of rC0C7-sc ligation. The cMyBP-C/α-actinin ratio in each lane was normalized to the cMyBP-C/α-actinin ratio in the untreated lane. > 90% ligation was achieved relative to uncut cMyBP-C when [rC0C7-sc] was > 5 μM. F) Addition of rC0C7-sc to HO myocytes without first treating with TEVp resulted in the appearance of a high MW band (> 250 kDa) consistent with formation of a branched (“Y”-shaped) cMyBP-C (right lane).