Analyses of calpain levels in the brain tissues of 263K-infected hamsters.

<p>A. Western blots of calpain in the infected and normal hamsters. B. Quantitative analyses of the gray numerical values of calpain. The average gray values were calculated from four infected hamsters or or four normal after normalized with that of individual β-actin and presented as mean ± SD. Statistical difference is illustrated as P<0.01. C. Western blots of calpain and tubulin in the brain tissues of 263K-infected hamsters on 0, 20, 40, 60 and 80 dpi. D. Quantitative analyses of the gray numerical values of calpain and tubulin vs that of individual β-actin. The average relative gray value is calculated from three independent blots and presented as mean ± S.D.</p

Immunofluorescent assays of MAP2 on PrP106–126 treated SK-N-SH cells.

<p>A. Immunofluorescence images of the cells were exposed to DMSO (upper), PrP106–126 (middle) or SCR (lower) for 6 h. The images of MAP2 (red), DAPI (blue) and merge are indicated above. B. Quantitative analysis of fluorescence intensity of MAP2 in the cells. MOD data each preparation is calculated from three independent images and presented as mean ± S.D. Statistical differences is illustrated as P<0.01.</p

Dynamic analysis of MAP2 and PrP^Sc in the brain tissues of normal and 263K-infected hamsters during incubation period.

<p>A. Western blots. Same amounts of individual brain homogenate were loaded in 6% or 12% SDS-PAGE. Various specific immunoblots were marked on the left and the time of post-inoculation are showed as days (d) at the bottom. B. Quantitative analysis of each gray numerical value of MAP2a/2b, MAP2c, 2d and PrP^Sc vs that of individual β-actin. The average relative gray value is calculated from three independent blots and presented as mean ± S.D.</p

Comparative analyses of the levels of MAP2 in brain tissues of normal and 263K-infected hamsters.

<p>A. Western blots. Same amounts of individual brain homogenate were loaded in 6% or 12% SDS-PAGE and various specific immunoblots were marked on the left side of the graphs. B. Quantitative analysis of each gray numerical value of MAP2a/2b, MAP2c, 2d, tubulin and total PrP vs that of individual β-actin. The average values were calculated from four individual infected hamsters or three individual normal hamsters and presented as mean ± SD. Statistical differences compared with controls were illustrated as P<0.05 or P<0.01. C. IHC assays of MAP2, total PrP and GFAP in cortex of normal and 263K-infected hamsters. The magnifications are ×20 in the left row and ×40 in the right row.</p

Analyses of the MAP2 levels in SK-N-SH cells exposed to PrP106–126.

<p>A. SK-N-SH cells were treated with 200 µM of PrP106–126, scrambled peptide PrP106–126 (SCR) or DMSO for 6 h and 12 h. The treated cells were photographed with a light microscope (×20). B. Cell viability after exposed to different concentrations of PrP106–126 or SCR. The average data of each preparation was calculated based on three independent experiments and represented as mean ± S.D. C. Western blots of MAP2a/2b, MAP2c, 2d and β-actin in SK-N-SH cells after treated with 200 µM of PrP106–126, SCR or DMSO for 6 h and 12 h. D. Quantitative analyses of each gray numerical value of MAP2a/2b, MAP2c, 2d vs that of individual β-actin. The average relative gray value is calculated from three independent blots and presented as mean ± S.D. Statistical differences compared with controls are illustrated as P<0.05 and P<0.01.</p

Analyses of calpain levels in PrP106–126 treated SK-N-SH cells.

<p>A. Western blots of calpain in cells exposed to DMSO, PrP106–126 or SCR. B. Quantitative analyses of the gray numerical values of calpain. The average relative gray value is calculated from three independent blots and presented as mean ± S.D. Statistical differences are illustrated as P<0.01. C. Western blots of MAP2 in the cells treated with PrP106–126 alone and PrP106–126 plus calpain inhibitor ALLN. D. Quantitative analyses of the gray numerical values of MAP2a/2b, MAP2d and MAP2c. The average relative gray value is calculated from three independent blots and presented as mean ± S.D. Statistical differences are illustrated as P<0.01.</p

Influences of overexpression of PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.

<p>A. Evaluation of the influence overexpressing PDI on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for PDI and PrP were reacted with individual antibodies. B. Cell viabilities assayed by CCK-8 kit. Each group is assigned to three repeating parallel units and measured with a spectrophotometer under 450 nm. The results are calculated from three independent tests and indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. C. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. D. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, pro-caspase3 vs that of individual β-actin. The results are calculated from three independent tests and presented as mean ± SD. Statistical differences compared with controls are illustrated. E. The relative densities of Bcl-2 and Bax specific bands normalized to β-actin are presented as ratio of Bax/Bcl-2. Statistical differences compared with controls are illustrated.</p

Inhibition of calpain activity reversed the PrP106–126-induced destruction on microtubule structures and cytotoxicity.

<p>A. SK-N-SH cells were treated with 200 µM of PrP 106–126 with or without calpain inhibitor ALLN for 12 h. Immunofluorescence images of cellular microtubule structure (upper) and morphological analyses of cells (lower) were shown. B. Cell viability after exposed to PrP106–126 with or without ALLN. The average data of each preparation was calculated based on three independent experiments and represented as mean ± S.D. Statistical differences are illustrated as P<0.05.</p

Analyses of the tubulin levels and microtubule structure in cells exposed to DMSO, PrP106–126 or SCR.

<p>A. Western blots with anti-α-tubulin mAb. B. Quantitative analyses of each gray numerical value of tubulin vs that of individual β-actin. The average relative gray value is calculated from three independent blots and presented as mean ± S.D. C. Immunofluorescence images of cellular microtubule structure.</p

Comparative analysis of the levels of PDIs in the brain tissues of normal and 263K-infected hamsters.

<p>A. Western blots. The infected hamsters were collected about 60 to 70 dpi. Same amounts of individual brain homogenate were loaded in 15% SDS-PAGE and immunoblotted with PrP specific mAb 3F4. PK+: with PK (50 µg/ml); PK-: without PK. B. Western blots of Grp78, Grp58, PDI and pro-caspase3. C. Quantitative analysis of each gray numerical value of Grp78, Grp58, PDI and pro-caspase3 vs that of individual β-actin. The average values are calculated from four infected hamsters or three normal hamsters and presented as mean ± SD. Statistical differences compared with controls are illustrated. D, Dynamic analyses of Grp78, Grp58, PDIs, pro-caspase3 and cleveage-caspase3 in the brain tissues of scrapie agent 263K-infected hamsters collected on 0, 20, 40 and 60 dpi during incubation period with Western blots. E. Log-fold changes of the gray values of Grp78, Grp58, PDIs, pro-caspase3 in the 263K-infected hamster’s brains collected on 20, 40 and 60 dpi vs that of 0 dpi.</p