Fig. 5. shRNA-mediated BIN1 knockdown restores youthful expression levels and localization patterns.

a Western blot of BIN1 expression in whole heart lysates from young and old mice probed with 2F11 (top) and 99D (bottom). Short and long exposures are displayed. Total ponceau was used for normalization. b Histogram showing normalized BIN1 levels in old relative to young for 2F11 and 99D (N = 6 biological replicates). c Quantitative RT-PCR analysis of pan-bin1, bin1, bin1 + 13, bin1 + 17, and bin1 + 13 + 17 transcripts for young and old myocytes are displayed normalized to gapdh (N = 3 per group, samples from each N were ran in triplicate through three separate PCR runs). d Representative Airyscan images of young (N = 3, n = 20), old (N = 3, n = 15), old shRNA-scrmb (N = 3, n = 16) and old shRNA-mBIN1 (N = 3, n = 19) myocytes immunostained against BIN1. e Western blot of BIN1 expression in whole heart lysates from: young, old, shRNA-mBIN1, and shRNA-scrmb transduced mice probed with 2F11. Short and long exposures are displayed. Total ponceau was used for normalization. f Histogram showing normalized BIN1 levels relative to young (N = 3 biological replicates). Statistical analysis was performed on data in (b) using unpaired two-tailed Student’s t-tests, on data in (c) using unpaired one-tailed Student’s t-tests, and on data in (f ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in (a, b, e, f ) are from JAX young, and young data in (c, d) are from NIA young myocytes. Note there was no significant difference in BIN1 expression when JAX and NIA young mice were compared (see Supplementary Fig. 1j–l). Data are presented as mean ± SEM. See Supplemental Information for full scans and number of technical replicates for western blots. Source data are provided in the Source Data file.