Fig 2: Hsp70 knockdown leads to increased protein carbonyl levels after oxidative stress. HT22 cells were transfected with siRNA against Hsp70 and/or Hsc70. 72 hr after transfection, cells were treated with 0.5 mM hydrogen peroxide for 0.5 hr, control cells were treated with PBS. (A) Cells were lysed 2 hr or 8 hr after hydrogen peroxide treatment and the lysates were used for protein carbonyl immunoblot (see Section 2). Representative immunoblots of protein carbonyls are shown. The two panels of Fig. 3A show the increase in protein carbonyl levels at 2 hr and at 8 hr after oxidative stress compared with the related controls. The columns are the means ± SD, n = 3–6, ***P < 0.005 vs. control, ****P < 0.001 vs. control, # P < 0.05 vs. w/o siRNA, ## P < 0.01 vs. scr. siRNA. (B) Cells were lysed with proteasome activity lysis buffer at 2 hr and 8 hr after hydrogen peroxide treatment, and used for measurement of ATP-dependent (26S) and ATP-independent (20S) proteasomal activity (see methods). Each column reports means ± SD, n = 3–6, *P < 0.05 vs. control.

Fig 3: Proteasomal degradation is increased in the presence of Hsp70. (A) HT22 cells were transfected with 25 nM scr. siRNA or 25 nM Hsp70 siRNA 24 hr after seeding. 72 hr after transfection, cells were incubated with growth medium, containing [35S]-labeled methionine and cysteine for 2 hr (for details see Section 2), washed and treated with 0.5 mM hydrogen peroxide or PBS for 0.5 hr. Afterwards, growth medium supplemented with 10 mM methionine/cysteine was added. Proteolytic degradation was measured at the desired time points in recovery after oxidative stress, as described in Section 2. The columns represent the proteolytic degradation (in % of scr. siRNA samples) at different time points after oxidative stress. They are the means ± SD, n = 7–9. ****P < 0.001 vs. control cells, ## P < 0.01 vs. scr. siRNA, # P < 0.05 vs. scr. siRNA. (B) Actin was incubated with hydrogen peroxide for 2 hr at 25 °C. Afterwards, hydrogen peroxide was removed by adding catalase. Oxidized actin (oxActin) was incubated with isolated 20S proteasome and with Hsp70 for 2 hr at 37 °C. Non-oxidized, native actin was used for comparison. Controls for each sample were incubated without the addition of 20S proteasome. The proteasomal degradation was measured using fluorescamine assay (for details see methods). The columns represent the proteasomal degradation, calculated as the difference between proteasome and control samples, respectively. They are the means ± SD, n = 3–4. **P < 0.01 vs. actin control, ### P < 0.005 vs. Hsp70 control. (C) Isolated 20S proteasome was incubated with Hsp70 and/or ATP and proteasomal activity was measured using the fluorogenic proteasome substrate suc-LLVY-MCA as described in methods. Columns are the means ± SD, n = 3.

Fig 4: Inducibility of Hsp70 after oxidative stress and heat shock. HT22 cells were either treated with 0.5 mM hydrogen peroxide for 0.5 hr or incubated for 1 hr at 42 °C. At the indicated time points after treatment, cells were lysed and immunoblots were performed. Representative immunoblots of Hsp70 and Hsc70 are shown. Hsp70 and Hsc70 concentrations were normalized to the density of GAPDH immunoblot or Ponceau staining. Columns are the means ± SD, n = 3–4, *P < 0.05 vs. control.

Fig 5: Interaction of Hsp70 with the 20 S proteasome is increased during recovery from oxidative stress. (A) HT22 cells were treated with 0.5 mM hydrogen peroxide for 0.5 hr. After recovery, cells were lysed using PBS. Lysates were used for immunoprecipitation of Hsp70 as described in methods. Immunoprecipitates (IP) were used for immunoblot against Hsp70, proteasomal subunit α4 and 20S core subunits. The columns display the ratios of co-immunoprecipitated proteasomal subunits per immunoprecipitated Hsp70 and are means ± SD, n = 3–4*P < 0.05 vs. control. (B) Hsp70 immunoprecipitation was performed from brain homogenates of three 18-month old 129/SV mice. The immunoprecipitates (IP) were used for immunoblots against Hsp70 and the proteasomal subunit α4. In panels C) and D) HT22 cells were transfected with HSPA1A-AC-GFP expression vector 24 hr after seeding. Further 24 hr later cells were incubated with 0.5 mM hydrogen peroxide for 0.5 hr. (C) At the indicated time points after treatment, cells were lysed using PBS, lysates were applied to a 10–40% sucrose gradient. Density gradient centrifugation was performed as described in methods. Fractions were analyzed for proteasomal activity and fractions with a high proteasomal activity were pooled and used for immunoblot against Hsp70 and 20S core subunits. The columns are the ratios between the amount of Hsp70 and the content of proteasomal subunits. The columns are means ± SD, n = 4–5, *P < 0.05 vs. control. Panel D) demonstrates the results of FRAP experiments 2 hr after hydrogen peroxide treatment. To inhibit the proteasome, experiments were performed in the presence of 2 µM LC. The data presented show the recovery time t1/2 after photobleaching in the presence of LC with the t1/2 of the non-LC-treated cells set as 100%. The insert shows the absolute recovery time t1/2 of all samples in seconds. The columns are the means ± SD, n = 4–5. *P < 0.05 vs. hydrogen peroxide control. Representative fluorescent images of one illustrative FRAP experiment are shown.