FIGURE 4:

CCT7 depletion causes redistribution of receptors in aggresomes. (A) HEK 293 cells stably expressing HA-TPβ transfected with CCT7 DsiRNA were fixed, permeabilized, and labeled with a rabbit anti-HA IgG and a mouse anti-GM130. Alexa Fluor 488–conjugated anti-rabbit IgG and Alexa Fluor 633–conjugated anti-mouse IgG were used as secondary antibodies. The fourth panel (d) represents a merge image of the blue (a), green (b), and red (c) signals. High degree of colocalization between the red and green signals appears in yellow. HEK 293 cells stably expressing HA-TPβ (B) or HA-β₂AR (D) were treated with control or CCT7 DsiRNAs. The cells were fixed, permeabilized, and labeled with mouse anti-HA IgG and stained with PROTEOSTAT aggresome dye. We used Alexa Fluor 633–conjugated anti-mouse IgG as secondary antibody. The third image on the right represents a merged image (c and f) of the green and red signals where the areas with high degree of colocalization between the green signal of the receptors (a and d) and red signal of the aggresome (b and e) appear yellow. Scale bars: 10 μm. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. (C, E) Mander’s colocalization coefficients represent the ratio of the green signal of the receptors overlapping the red signal of the aggresome and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.