Morphological assays of the effects of various recombinant TPPP proteins on the fibril formation of synthetic PrP106–126 <i>in vitro</i> with a transmission electronic microscopy.

<p>0.5 mg/ml synthetic peptide PrP106–126 was incubated in the absence (A) or presence of TPPP1–219 (B), TPPP50–219 (C) and TPPP100–219 (D) at 37°C for 72 h, respectively. The molar ratio of PrP106–126 to TPPP was 2∶1. Magnification was 97,000×. Scale bar represented 100 nm.</p

Analyses of cell viabilities receiving various plasmids expressing PrPs and/or TPPP.

<p>The cell viabilities of HeLa (A) and SHSY5Y (B) cells transfected with various plasmids were measured by a commercially Cell Counting Kit 24 and 48 h after transfection. Cells treated with colchicines (10 µM) were used as controls. The average data of each preparation was calculated based on three independent experiments and represented as mean ± S.D.</p

Protein purification of various recombinant TPPP and PrP proteins.

<p>A. 15% SDS-PAGE assay for various purified recombinant TPPP proteins. M: standard molecular weights of protein. B. Western blot assay for various recombinant TPPP proteins with anti-TPPP mAb. C. Western blot assay for various recombinant PrP proteins with different antibodies. His-PrP(23–231) and His-PrP(90–231) were blotted with anti-PrP pAb. GST, GST-PrP(106–126) and GST-PrP(23–90) were blotted with anti-GST mAb. Protein molecular markers are shown on the left.</p

Molecular interactions between the expressed PrP and TPPP in HeLa cells.

<p>A. Western blot analyses of the expressions of PrP (left panel) and TPPP (right panel) in the HeLa cells transfected with or without PrP or TPPP expressing plasmid. Cells were harvested 48 h post-transfection and cell lysates were separated onto 12% SDS-PAGE. B. Co-immunoprecipitation assays of the expressed PrP and TPPP in HeLa cells. The PrP-TPPP complexes in cell lysates were captured by anti-TPPP pAb and blotted by anti-PrP mAb (left panel), or captured by anti-PrP mAb and blotted by anti-TPPP pAb (right panel). Protein molecular markers are shown on the right.</p

Molecular interactions between various recombinant TPPP and PrP.

<p>A. GST pull-down assay of various recombinant TPPP with His-PrP23–231. The TPPP-PrP complexes were precipitated with glutathione agarose beads. The bound PrP was detected by PrP specific 3F4 mAb. B. Co-immunoprecipitation assay of various TPPPs with His-PrP23–231. The TPPP-PrP complexes were precipitated by anti-TPPP pAb and the bound PrP was detected with PrP specific 3F4 mAb. C. His pull-down assay of various His-tagged PrPs with GST-TPPP1–219. The TPPP-PrP complexes were precipitated with Ni-NTA agarose beads. The bound TPPP was detected by anti-TPPP pAb. D. Co-immunoprecipitation assay of various His-tagged PrPs with GST-TPPP1–219. The TPPP-PrP complexes were precipitated by anti-TPPP pAb and the bound PrP was detected with PrP specific 3F4 mAb. E. Co-immunoprecipitation assay of various GST-tagged PrP with GST-TPPP1–219. The TPPP-PrP complexes were precipitated by anti-TPPP mAb and the bound PrP was detected with anti-PrP pAb. F. Co-immunoprecipitation assay of various PrPs in the context of full-length with TPPP1–219. The TPPP-PrP complexes were captured by anti-PrP pAb and the bound TPPP was detected with anti-TPPP mAb. PrP-input represents the purified PrP23–231 and TPPP-input represents the purified GST-TPPP1–219, directly loaded as controls of Western blots. Protein molecular markers are shown on the left.</p

Morphological analyses of the influences of TPPP and PrP on the structures of microtubule in HeLa cells by a confocal microscopy.

<p>The structures of microtubules of the cells treated with various agents were monitored 48 h post-transfection. Panel A: colchicines (10 µM). Panel B: pcDNA3.1-CytoPrP. Panel C: pcDNA3.1-CytoPrP and pcDNA3.1-TPPP. Panel D: pcDNA3.1. Panel E: pcDNA3.1-PG5. Panel F: pcDNA3.1-TPPP.</p

Comparative analyses of the levels of TPPP and PrP in the brain tissues of normal and scrapie agent 263K-infected hamsters collected at the moribund stage.

<p>A. Western blot assays for the endogenous TPPP and PrP with anti-TPPP pAb and mAb 3F4, respectively. Same amounts of individual brain homogenate were loaded in 12% SDS-PAGE. The scrapie infected as well as normal hamsters were indicated on the top of the graphs. The specific immunoblots of TPPP, PrP and β-actin were indicated on the left and molecular mass markers were indicated to the right. B. Quantitative analyses of each average grey value of the preparations after equilibrated with that of individual β-actin. The average grey values were calculated from four scrapie infected hamsters and four normal ones and presented as mean ± SD. Statistical differences compared with normal controls were illustrated as P<0.05.</p