Figure 3. Thermal stress induces both Jak2 degradation and aggregation.

(A) Equal amounts of isolated PBMCs were incubated at 37°C and 40°C, respectively, for 4 h. The cells were either lysed in 1% SDS and analyzed in western blot (TCL) or lysed in 1% NP-40; the lysates were centrifuged (500 g), the supernatants were collected and the pellets were dissolved in 1% SDS. Equal aliquots were analyzed by western blotting (WB) using anti-Jak2, anti-Jak3 and anti-STAT5b antibodies. (B) Equal amounts of isolated PBMCs were incubated at 40°C for the indicated times, lysed in 1% NP-40, and analyzed as in Fig. 3A (WB) using anti-Jak2 and anti-STAT5b antibodies. For the right lanes in the four panels, the incubation was continued for 3 h at 37°C. (C) Equal amounts of Hek293-TR cells were lysed in 1% Triton X-100, the lysates were clarified by centrifugation, and incubated at 37°C and 40°C, respectively, for 4 h. Next, the lysates were centrifuged at 500 g, and equal aliquots of supernatant and pellet were analyzed by western blotting (WB), using anti-Jak2 antibody. The upper band is an unspecific background band. (D) γ2A Jak2 −/− cells, transfected with GFP-Jak2, were incubated at 37 and 40°C for 4 h and fixed in formaldehyde. Representative pictures are shown. Fluorescence was visualized with a confocal microscope. Bar, 20 µm. All data in this figure are representative of three independent experiments.