Cavin4b is recruited to Bin1b-positive tubules in BHK cells. (A–D) Live confocal imaging of BHK cells cotransfected with: EGFP-Bin1b/Cavin4b-mCherry (A), EGFP-BIN1/CAVIN4-mCherry (B), EGFP-Bin1b/Cavin4b(PRDmt)-mCherry (C), or EGFP-Bin1b/Cavin4a-mCherry (D). Colocalization was analyzed by line scan (as indicated, inset = line scan area) showing pixel intensity over the line distance (far right panel). Images representative of n = 6 cells/group and three replicates. Scale bars: 10 µm (insets, 5 µm). (E) Time-lapse confocal imaging of BHK cells cotransfected with EGFP-Bin1b/mCherry or EGFP-Bin1b/Cavin4b-mCherry. Each frame was color coded and superimposed into a single image. Arrowheads indicate dynamic Bin1 tubules captured during time lapse. Scale bars: 10 µm. (F) Scatter plot quantitation of number of Bin1b-positive tubules (normalized to cell size) captured during live cell imaging (6 min; n = 5 cells; nested two-tailed t test). **, P ≤ 0.01. Error bars represent mean ± SD.