FIGURE 8.

TfR1 association with the chaperone Hsc70 increases upon c-Abl inhibition by imatinib. a and b, following imatinib treatment, clone 9 cells were subjected to coimmunoprecipitation using an antibody against TfR1 (a) or Hsc70 (b) and analyzed by Western blot. Note that binding between the two proteins increased markedly after c-Abl inhibition. a′ and b′, quantitation of three independent experiments as described in a and b. Data are presented as mean ± S.E.; **, p < 0.01. c–f, clone 9 cells expressing Hsc70-GFP or LAMP1-GFP were treated with vehicle control (c and e) or 20 μg/ml imatinib (d and f) for 2 h and subsequently analyzed by immunofluorescence microscopy using an antibody against TfR1 (c′ and d′) or LAMP1 (e′ and f′) to assess colocalization between the proteins tested. Indeed, Hsc70 colocalized with TfR1 and LAMP1 at the lysosome after imatinib treatment. Scale bar, 10 μm. g, quantitation of the morphology-based experiments shown in c and d showing a marked increase in overlap between Hsc70-GFP- and TfR1 antibody-stained compartments following control or imatinib treatments. (Control, 0.6 ± 0.006%, n = 15 cells; imatinib: 14.1 ± 0.09, n = 20 cells.) h, quantitation of the morphology-based experiments as described in e and f showing LAMP1-GFP- and anti-Hsc70-stained compartments following control or imatinib treatments. A substantial increase in localization of the chaperone to the lysosome compartment is observed. (Control, 5.4 ± 0.031%, n = 16 cells; imatinib, 19.9 ± 0.12, n = 25 cells.) **, p < 0.01.