Figure 5.
Characterization of actin-bundling ability of TPRN. All TPRN fragments and FL TPRN have a TRX tag at the N-terminus. (A) Actin filaments in the presence of a TRX tag, FL TPRN, or fragments of TPRN. Actin (3 μM) was incubated with 3 μM TRX, 0.1 μM FL TPRN, 0.15 μM TPRN1–400, 0.5 μM TPRN1–300, 0.75 μM TPRN1–170, or 1.5 μM TPRN301–749 at RT for 1.5 h, followed by labeling with fluorescent FITC-phalloidin. The experiment was performed more than three times, yielding consistent results. Scale bars: 5 μm. (B–F) Low-speed co-sedimentation assays were used to determine the cross-linking activity of TPRN fragments. Actin (3 μM) was incubated with buffer or 0.2 μM TPRN1–400 (B), 0.5 μM TPRN1–300 (C), 0.75 μM TPRN1–170 (D), 3 μM TPRN301–749 (E), or 9 μM TPRN1–63 (F) at RT for 1.5 h, followed by centrifugation. Equivalent amounts of supernatant (S) and pellet (P) were separated using SDS-PAGE and stained with Coomassie blue. Arrows with numbers on the right-hand side of each gel indicate corresponding TPRN fragment length in aa used in each experiment. All experiments were repeated at least three times. (G) Actin was incubated with varying amounts of FL TPRN or fragments of TPRN. Then, low-speed co-sedimentation assays were performed. The percentage of actin in the pellet was quantified (n ≥ 3). Data are represented as the mean ± SEM. (H) The TRX-TPRN schematics illustrating actin-bundling activities of various TPRN fragments indicated by a bracket based on co-sedimentation data showing that bundling relies on the N-terminal part of TPRN, while all TPRN fragments can bind actin and oligomerize. Source data are available for this figure: SourceData F5.

Characterization of actin-bundling ability of TPRN. All TPRN fragments and FL TPRN have a TRX tag at the N-terminus. (A) Actin filaments in the presence of a TRX tag, FL TPRN, or fragments of TPRN. Actin (3 μM) was incubated with 3 μM TRX, 0.1 μM FL TPRN, 0.15 μM TPRN1–400, 0.5 μM TPRN1–300, 0.75 μM TPRN1–170, or 1.5 μM TPRN301–749 at RT for 1.5 h, followed by labeling with fluorescent FITC-phalloidin. The experiment was performed more than three times, yielding consistent results. Scale bars: 5 μm. (B–F) Low-speed co-sedimentation assays were used to determine the cross-linking activity of TPRN fragments. Actin (3 μM) was incubated with buffer or 0.2 μM TPRN1–400 (B), 0.5 μM TPRN1–300 (C), 0.75 μM TPRN1–170 (D), 3 μM TPRN301–749 (E), or 9 μM TPRN1–63 (F) at RT for 1.5 h, followed by centrifugation. Equivalent amounts of supernatant (S) and pellet (P) were separated using SDS-PAGE and stained with Coomassie blue. Arrows with numbers on the right-hand side of each gel indicate corresponding TPRN fragment length in aa used in each experiment. All experiments were repeated at least three times. (G) Actin was incubated with varying amounts of FL TPRN or fragments of TPRN. Then, low-speed co-sedimentation assays were performed. The percentage of actin in the pellet was quantified (n ≥ 3). Data are represented as the mean ± SEM. (H) The TRX-TPRN schematics illustrating actin-bundling activities of various TPRN fragments indicated by a bracket based on co-sedimentation data showing that bundling relies on the N-terminal part of TPRN, while all TPRN fragments can bind actin and oligomerize. Source data are available for this figure: SourceData F5.

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