Figure 1.
Effect of stressors on Jak2 protein levels in various cell types.
(A) Hek293-TR, Chinese hamster lung and PBM cells were incubated for 3 h, as indicated. The cells were lysed in 1% Triton X-100 and equal aliquots of the lysates were analyzed by western blotting (WB) for endogenous Jak2, using anti-Jak2 antibody. The antibody recognized a-specific bands in lysates of HekTR (above Jak2) and CHL (below Jak2). (B) In the presence of 20 µg/ml cycloheximide (CHX) Chinese hamster cells were transiently transfected with Jak2 and incubated at either 37 or 40°C. The cells were lysed in 1% SDS and equal aliquots were analyzed (WB) using anti-Jak2 and anti-actin antibodies. (C) Equal amounts of isolated PBMCs were incubated with or without 20 µM MG-132 at 37 or 40°C for 4 h. The cells were lysed in 1% SDS and equal amounts were analyzed (WB) using anti-Jak2 antibody. (D) Effect of Epoxomicin on Jak2 protein levels in PBMCs. Equal amounts of isolated PBMCs were incubated with or without10 µM Epoxomicin at 37 or 40°C for 4 h. The cells were lysed in 1% SDS and equal amounts were analyzed (WB) using anti-Jak2 antibody. The data in (A, B, C, D) are representative of three independent experiments. (E) Quantification of (D). The ratio of Jak2 to actin signals (western blot) were calculated. The data represent the mean of three independent experiments ± SEM.
Figure 2.
JAK/STAT signaling response is diminished under elevated temperature.
(A) Mouse 3T3-F442A preadipocytes were incubated at 37 or 40°C for 4 h and then treated with the indicated concentrations of GH for 10 min. The cells were lysed in 1% SDS and equal amounts of lysate were analyzed by western blotting (WB), using anti-Jak2, anti STAT5b and anti-actin antibodies. (B) Quantification of Fig. 2A. At each point the ratio of the lower band to the total STAT5b signal was calculated (100 ng/ml GH at 37°C was set 100%; 0 ng/ml was set 0%). The data represent the mean of four independent experiments SEM. (circles, 37°C; squares, 40°C).
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.
Figure 4.
Kinase inactive Jak2 is degraded but not aggregated.
γ2A Jak2 −/− were transiently transfected with Jak2-K882E or wild type Jak2, respectively. Equal amounts of cells were incubated at 37 and 40°C, respectively, for the indicated times. The cells were lysed, fractionated and analyzed as in Fig. 3B. The data in this figure are representative of three independent experiments.