An interaction between Bin1b and Cavin4b contributes to tubule formation. (A and B) Ultrastructural analysis of 5-dpf WT (A) and cavin4^−/− (B) zebrafish embryos. Arrowheads indicate caveolae in WT embryo. Scale bars: 1 µm. (C and D) Live confocal imaging of BHK cells cotransfected with: EGFP-SNX8/Cavin4b-mCherry (C, n = 5 cells imaged) or EGFP-Bin1b/mCherry (D, n = 3 cells imaged each in five independent experiments). Colocalization was analyzed by line scan (as indicated; inset shows line scan area) showing pixel intensity over the line distance (far right panel). Scale bars: 10 µm. (E and F) Scatter plot quantitation of tubule area (E) and average Feret’s diameter (F) of Bin1b-positive tubules in the presence of Cavin4b-mCherry or mCherry reporter during time-lapse imaging over 6 min (n = 5 cells analyzed). Tubule area was normalized to cell size. For E, nested two-tailed t test, area normalized to cell size. For F, two-tailed t test. *, P ≤ 0.05; **, P ≤ 0.01. Error bars represent mean ± SD. (G) Uncropped gels from Fig. 6 B (shown as a multichannel image). Pull-down using GFPtrap with a maltose binding protein tag with in-gel fluorescence detection after semi-denaturing PAGE. Cavin4b-mCherry was coexpressed with Cavin4b-EGFP (positive control), EGFP-Bin1a, EGFP-Bin1b, or EGFP (reporter only negative control) in BHK cells. Protein in starting lysate is shown on left, pull-down using GFPtrap with a maltose binding protein tag is shown on right. Proteins in pull-down fraction appear as a doublet due to binding to maltose. M, molecular weight marker. (H) Schematic of zebrafish Cavin4b protein domains (HR, helical region). Alignment between PRD within DR3 of Cavin4b and the SH3-binding region of CHIKV is shown; proline residues are in red, and positively charged amino acids are highlighted in yellow. (I) Direct association of Bin1b SH3 domain with CHIKV peptide as measured by ITC.