Fig. 2: Directed severing of HaloTag-TEV titin.

a Following treatment with TEV protease, the HaloTag-TEV titin is expected to result in at least two fragments: A-M-band fragment (~2.2 MDa) and the Z-disk/I-band fragment(s), which can show a range of molecular weights depending on the specific titin isoform (represented by asterisk and brackets in the titin diagram). b Digestion of cardiac titin is tracked by 1.8% polyacrylamide SDS-PAGE electrophoresis and Coomassie staining (a representative image of 20 experiments showing the same result). Wild-type titin is not affected by incubation with TEV protease, so typical bands corresponding to full size N2BA/N2B isoforms and to the T2 fragment or the Cronos isoform are observed in both lanes. In contrast, digestion of HaloTag-TEV titin results in a main band at 2.2 MDa, corresponding to the A-M-band fragment, and two extra bands at 0.8 and 1.0 MDa that are assigned to the I-band portions of the N2B and N2BA cardiac isoforms, respectively. c Passive stiffness of skinned bundles of psoas myofibers isolated from homozygous HaloTag-TEV titin mice (H/H). During mechanical testing, myofibers were stretched in steps to increasing sarcomere lengths, and the resulting passive force was recorded. Force was first measured in relaxing buffer, then myofibers were incubated with TEV for 30 min at slack length (2.2 μm), and the measurement was repeated. Inset: control experiment with wild-type fibers. d The drop in passive force that results from incubation of HaloTag-TEV titin fibers with TEV protease is time-dependent (in these experiments, H/H fibers were returned to slack length between consecutive mechanical tests at 10, 20, 40, and 120 min; data at 10 and 40 min are not presented for clarity). After 120 min, high ionic strength KCl/KI buffer was added to extract titin. To average several experiments (n = 6), force values are normalized to the value at 3.4 μm SL before treatment with TEV. Error bars represent SEM. Source data are provided as a Source Data file. e Mechanical testing at long sarcomere lengths. Left: Experimental protocol. Right: Results from six independent experiments using ~150-μm-diameter skinned psoas fibers.