Shr3 specifically associates with Gap1 truncations. Strain FGY135 (gap1Δ shr3Δ) expressing SHR3-GFP (pAR46) and carrying plasmids gap1-8TM (pIM4), gap1-10TM (pIM5), hxt1-8TM (pIM37), or hxt1-10TM (pIM38; schematically represented) was induced with 2% galactose for 1 h. Extracts were prepared and Shr3-GFP was purified with GFP-trap agarose beads in the presence of 0.8% n-dodecyl-β-D-maltoside (DDM) as described (Methods and materials). Bound proteins, resolved by SDS-PAGE, were analyzed by immunoblot using α-HA, α-GFP, and α-Dpm1 antibodies. The input (I; lanes 1–4) correspond to 1.8% of extracts incubated with the GFP-trap beads. Extracts were separately analyzed (lower panel) to analyze levels of Shr3-GFP. The signal intensities of the immunoreactive forms of Cub-GST-6xHA constructs were quantified (right panel); the mean values of the Bound% are plotted with error bars showing standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData FS6.