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. 2005 Oct 19;25(42):9735–9745. doi: 10.1523/JNEUROSCI.1912-05.2005

Figure 4.

Figure 4.

The removal of HSP70 from MEx reduces its survival-promoting activity. Coomassie-stained PVDF membranes (A and B) and corresponding Western blots (A′ and B′) resulting from ion exchange chromatography with DE-52 medium (Whatman) are shown. The pink arrow indicates the 64 kDa marker on each membrane. Lane 1 shows the flow-through, and lane 3 shows the wash fraction, both of which show no immunoreactivity for HSP70, indicating that HSP70 is completely bound to the column. Fractions 5-31 show the elutions by a stepwise salt gradient of 100 mm (5-15), 125 mm (17-19), and 150 mm (23-31). Further increased salt concentrations (300 mm) were used to elute the remainder of proteins contained in MEx (data not shown). Western blots of the elution fractions indicate the location of HSP70 (top band) and actin (bottom band; pink arrow indicates 65 kDa marker). The fractions containing HSP70 were pooled, dialyzed into ATP column buffer B, and subjected to affinity chromatography on a C-8-linked ATP agarose column (Sigma). Coomassie-stained PVDF indicates intact HSP70 eluted in fractions 11-19 (C; lane 15). Corresponding Western blot (C′) indicates that the protein is immunoreactive to Hsp70. After chromatography, the fractions from ion-exchange and noneluted ATP column were pooled (D; sample R for removal). As a control for the removal process, after chromatography, the fractions from ion-exchange and noneluted ATP column were pooled together with the ATP elution fractions containing the HSP70 bound to the column (D; sample C for control). Pooled samples are dialyzed in PBS and lyophilized to concentrate them. Western blot and Coomassie analysis were performed to confirm the reduction of HSP70 in MEx (D). Western blot analysis indicates a diminished amount of HSP70 in the depleted sample (R) compared with the control (C). Western blot for actin and a Coomassie-stained membranes were used for loading controls and comparison of extract integrity. E, Motoneurons were cultured either with trophic support (MEx), without trophic support (no MEx), with the control MEx (Control), or MEx in which both Hsc and Hsp70 were depleted from the extract (Removal). Those motoneurons treated with the depleted extract showed a significant decrease in the number of surviving cells compared with both MEx and the control extract. Results are presented as mean + SEM; n = 4 independent experiments. Statistical significance was determined using ANOVA followed by Tukey-Kramer post hoc test. *p ≤ 0.05 compared with MEx; **p ≤ 0.01 compared with control; ***p ≤ 0.001 compared with MEx and control.