Figure 6

HSP70 inhibits translocation of NF-κB and degradation of IκBα. Heat shock protein (HSP)70 inhibits tumour necrosis factor (TNF)-α induced nuclear translocation of nuclear factor-κB (NF-κB) and degradation of IκBα in fibroblast-like synoviocytes (FLSs) detected by Western blot. (a) Human rheumatoid arthritis (RA) FLSs were incubated with HSP70 at 0.1 to 10 μg/ml for 1 hour, and the FLSs were washed and exposed to TNF-α (20 ng/ml) for 30 minutes. Nuclear or cytoplasmic lysates were immunoblotted with anti-P65, anti-PCNA (proliferating cellular nuclear antigen), or anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase). PCNA and GAPDH served as controls for nuclear and cytoplasmic proteins. The levels of p65, GAPDH and PCNA were estimated by densitometry. Shown in the left panels are representative Western blots, and shown in the right panels are the means ± standard deviation of three independent experiments. *P < 0.05 versus the TNF-α group. (b) Human RA FLSs were incubated with HSP70 at 0.1 to 10 μg/ml for 1 hour. Then, the FLSs were washed and exposed to TNF-α (20 ng/ml) for 20 minutes. Cytoplasmic lysates were immunoblotted with IκBα or anti-tubulin. Tubulin served as a control for cytoplasmic protein. The levels of IκBα and tubulin were estimated by densitometry. Shown in the left panel is a representative Western blot, and shown in the right panel are the means ± standard deviation of three independent experiments. *P < 0.05 versus the TNF-α group.