Panel A: The plasmid vector map represents the genetic construct p E T 52 b: m A164-167 A L F A 10xHis, which has a total size of 6421 base pairs (bp). The circular map identifies various functional components labeled with arrows and text boxes, including the lacI gene (purple arrow), a lac operator, and a lacI promoter positioned upstream of the main expression cassette. The cassette itself contains a T 7 promoter and a ribosome binding site (R B S) followed by the specific titin immunoglobulin domains A164 (red), A165 (red), A166 (white), and A167 (white). For protein purification and detection, the map shows several tag sequences, including a Strep-Tag 2, an H R V 3 C site, an A L F A-tag, a thrombin site, and a 10xHis tag, terminating at a T 7 terminator. The backbone includes additional regulatory and selection elements. Panel B: Protein Pull-Down Assay shows a Coomassie-stained S D S-P A G E gel used to analyze the interaction between recombinant titin domains and muscle lysates. The left-most lane contains a molecular weight marker with labels ranging from 25 to 250 kilodaltons. The subsequent lanes show various combinations of the recombinant protein rA164-167-A L F A, Gastrocnemius (Gast) lysate, and Left Ventricle (L V) lysate. A very thick, prominent band is visible at approximately 55 kilodaltons in all lanes where the recombinant protein was added, corresponding to its predicted molecular weight. Panel C: Protein Identity Table lists the candidate protein interaction partners identified from the pull-down assay based on their molecular weights. The first column, Molecular Weight, lists three categories: 250 kilodaltons, 100 kilodaltons, and 30 kilodaltons. The second column, Protein Identity, correlates these weights to specific proteins: Myosin-4 and Myosin-1 at 250 kilodaltons; S E R C A 1 and S E R C A 2 at 100 kilodaltons; and A N T 1 and A N T 2 at 30 kilodaltons. Panel D: L V A N T 1, 2 Expression. This panel quantifies the protein expression level of A N T 1/2 in the Left Ventricle (L V). The vertical axis represents the ratio of A N T 1/2/G A P D H, ranging from 0.000 to 0.020 with increments of 0.005. The W T group (gray bar) has a mean value of approximately 0.011, while the Ttn Delta A164-167 group (blue bar) is slightly higher with a mean of approximately 0.013. Below the bar graph, a western blot shows the bands for G A P D H (top) and A N T 1/2 (bottom) for both genotypes. A bracket labeled ns indicates that the difference in A N T 1/2 expression in cardiac tissue is not statistically significant. Panel E: E D L A N T 1/2 Expression. This panel quantifies the expression of A N T 1, 2 in the Extensor Digitorum Longus (E D L) muscle. The vertical axis represents the A N T 1/2/G A P D H ratio, ranging from 0.0 to 1.0 with increments of 0.2. The W T group (gray bar) averages roughly 0.44, and the Ttn Delta A164-167 group (blue bar) averages roughly 0.54. Similar to the cardiac data, the western blot bands for G A P D H and A N T 1/2 are shown below the graph. The bracket labeled ns confirms that there is also no statistically significant difference in the expression of these proteins in skeletal muscle between the two groups. All values are approximate.
Pulldown assays to probe for potential binding partners of the titin A164–167 segment. (A) Map of the plasmid used to produce the recombinant titin A164–167 bait protein. (B) Representative gel image from a pulldown assay in which the recombinant ALFA-tagged A164–167 protein (rA164–167-ALFA) was used to probe gastrocnemius (gast) or LV tissue lysates. The boxed areas were cut from the gel and analyzed by LC-MS/MS to identify the main protein components. (C) Table of the bands tested by mass spectrometry and the top protein hits. (D) Western blot quantification of ANT1/2 levels normalized to GAPDH in LV tissue lysate. (E) Western blot quantification of ANT1/2 levels normalized to GAPDH in EDL tissue lysate. Statistical significance for D and E was determined by an unpaired t test. Tissue lysates from 8 WT and 8 TtnΔA164–167 were used. Source data are available for this figure: SourceData FS2.