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. 2019 Aug 29;47:329–340. doi: 10.1016/j.ebiom.2019.08.045

Fig. 4.

Fig. 4

cTnIAAb binds to α-enolase (ENO1) in myocardial cell membrane.

a. The concentrated purified protein from BCG823 cell lysates through cTnImAb1-dextran affinity chromatography was subjected to SDS-PAGE followed by Coomassie blue staining, which revealed one single band.

b. The purified protein from BCG823 cell lysates specifically bound to ENO1 mAb, but could not bind to cTnImAb1 again.

Left: Western blotting showing that the purified protein from BCG823 cell lysates through cTnImAb1-dextran affinity chromatography could not interact with cTnImAb1 again.

Middle: Western blotting showing that the purified protein from BCG823 cell lysates through cTnImAb1-dextran affinity chromatography could specifically bind to ENO1 mAb (51 KDa). Right: Western blotting showing that the non-reduced protein lysates from mouse cardiomyocytes interacted with cTnImAb1 at both 28 kDa and 51 kDa, while the reduced protein lysates interacted with cTnImAb1 only at 28 kDa.

c. Immunoblot analysis showed that cTnImAb1 and anti-ENO1 pAb bound to the immunoprecipitation-purified endogenous dimeric ENO1 from cardiomyocytes.

Far Left: Mouse cTnImAb1 strongly interacted with the unreduced ENO1 dimer (line 6, ~98 kDa) immunoprecipitated by rabbit ENO1 mAb155102, while it weakly interacted with the reduced endogenous ENO1 (line 3, ~47 kDa). Mouse cTnImAb1 interacted with myocardial cTnI (lines 2 and 5, ~28 kDa) as well as the unreduced input protein sample (line 2, ~98 kDa).

Left: Mouse ENO1 pAb strongly interacted with the unreduced ENO1 dimer (line 6, ~98 kDa) immunoprecipitated by rabbit ENO1 mAb155102, while it weakly interacted with the reduced endogenous ENO1 (line 3, ~47 kDa). Mouse ENO1 pAb interacted with myocardial ENOI (lines 2 and 5, ~47 kDa) as well as the unreduced input protein sample (line 2, ~98 kDa).

Right: Rabbit ENO1 mAb155955 interacted with ENO1 in the input protein sample (lines 2 and 5) as well as the immunoprecipitated endogenous ENO1 by rabbit ENO1 mAb155102 (lines 3 and 6). Rabbit ENO1 mAb155102 also bound to anti rabbit IgG (lines 4 and 7).

Far Right: Mouse actin mAb interacted with the actin in the input protein sample (lines 2 and 5). All images are representative of at least 3 independent iterations.

d. Identification of FLAG-tagged recombinant wild-type and C389A, C357A, C339A, C119A ENO1 protein expressed in 293 T cells.

e. The reaction of recombinant ENO1 protein and 4 point-mutated ENO1 proteins with cTnImAb1 and Anti-ENO1 (non-reducing sample) respectively. Results shown are representative immunoblot analysis of three independent co-immunoprecipitation analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)