TanGIBLE recognizes mislocalized TMD protein more than BAG6 N200 or N465 fragments. (A) In relation to the Fig. 1, schematic of the domain structure of the N-terminal 465 residue region of human BAG6. Domain II contains DUF3538 (276–392, indicated by a yellow square). Low evolutionarily conserved sequences are indicated by gray lines. The amino acid sequence of BAG6 N465 was analyzed using PONDR. The stretches of the UBL sequence are indicated by blue boxes, the regions of the BUILD sequence are indicated by red boxes, and DUF3538 and other evolutionarily conserved regions in Domain II are indicated by yellow boxes. The results from two different predictor package, VL-XT (red line) and VL3 (blue line), are shown. (B) In relation to Fig. 2, improved detection of mislocalized IL-2Rα with TanGIBLE. T7-tagged BAG6 derivative fragments were immunoprecipitated (IP) and blotted using anti-Flag antibody to examine the interaction with Flag-IL-2Rα ΔSS. A N200-6S fragment (see Fig. 2 B) was used as the negative control. α-Tubulin was used as the loading control. (C) TanGIBLE co-precipitates more IL-2Rα ΔSS protein than BAG6 N200 and N465 fragments. After transfection with expression vectors encoding T7-IL-2Rα ΔSS and Flag-tagged BAG6 derivative fragments, anti-Flag immunoprecipitates were blotted with anti-T7 or anti-Flag antibodies. (D) Quantification of IL-2Rα ΔSS signals co-precipitated with Flag-tagged TanGIBLE. The graph represents the mean ± SD calculated from three independent biological replicates. P values were calculated using Dunnett’s multiple comparisons test between N200, N465, and TanGIBLE. This statistical analysis was performed using the computing environment R version 3.5.1. Source data are available for this figure: SourceData FS1.