Table 1.
Characteristics of the cell lines and cell extracts used throughout this study.
Figure 1.
CCT/TRiC quantities in cancer cell lines.
The amount of CCT/TRiC in 18 cancer and a normal (MRC5-SV2) cell lines extracts, prepared as described in the experimental procedures section, a non-cancer liver homogenate (HNCL) and rabbit reticulocyte lysate (RRL) was determined by comparing CCTα intensity measured for cell extracts diluted 4 and 16 fold to that of known amounts of CCTα (100, 50, 25, 12.5 µg/ml) migrated on the same 12% SDS polyacrylamide gel by Western blotting. The average amounts of CCT/TRiC in the different extracts are expressed as a function of the total proteins content of the extracts.
Figure 2.
CCT/TRiC folding activity in cancer cell lines extracts.
CCT/TRiC folding activity in the 18 cancer cell lines, the normal cell line MRC5-SV2 and the human non-cancerous liver was assayed using [35S]-labeled, denatured β-actin folding as described in the experimental procedures section. The reaction products were analyzed on 6% non-denaturing polyacrylamide gels. CCT/TRiC specific activity, derived from the quantification of β-actin bands intensities is expressed as a function of total protein concentration. The images, recorded using a phosphorimager for [35S]-labeled actin refolding reactions in folding buffer, RRL, MIA-PaCa-2 and HeLa cell extracts resolved on a native 6% polyacrylamide gel, are shown in the inset.
Figure 3.
CCT/TriC activity modulators in cancer cell lines extracts.
The amount of (A) Hop/p60, (B) PhLP3 and (C) PFD in 18 cancer and a normal (MRC5-SV2) cell lines extracts, a non-cancer liver homogenate (HNCL) and rabbit reticulocyte lysate (RRL) was determined by comparing the chemiluminescence signal recorded for Hop/p60, PhLP3 and PFD in cell extracts diluted 4 and 16 fold to that of known amounts of the proteins on the same Western blots.
Figure 4.
CCT/TriC, Hop/p60 or PhLP3 immunodepletion affects actin refolding.
CCT/TRiC folding activity in (A) MIA-PaCa-2 and (C) OVCAR-8 cell extracts before and after CCT/TriC, Hop/p60 or PhLP3 immunodepletion was assayed using [35S]-labeled, denatured β-actin. The protein concentration of the MIA-PaCa-2 and OVCAR-8 cell extracts was 2.5 and 15 mg/ml, respectively. Native β-actin band intensity was measured using a PhosphorImager. The amount of native β-actin measured after immunodepletion is expressed as a fraction of the amount measured for the cell extracts before immunodepletion in (B) MIA-PaCa-2 and (D) OVCAR-8 cell extracts.
Figure 5.
Hsc/p 70 expression in cancer cell lines extracts.
The amount of Hsc/p70 in 18 cancer and a normal (MRC5-SV2) cell lines extracts, a liver homogenate (HNCL) and rabbit reticulocyte lysate (RRL) was determined by comparing the chemiluminescence signal recorded for Hsc/p70 in cell extracts diluted 4 and 16 fold to that of known amounts of the protein on the same Western blots. The average amounts of Hsc/p70 in the different extracts are expressed as a function of the total proteins content of the extracts.
Figure 6.
Hsp/c70 immunodepletion affects CCT/TRiC-mediated β-actin folding.
CCT/TRiC-mediated labeled, denatured β-actin folding in (A) MIA-PaCa-2 and OVCAR-8 cell extracts before and after Hsc/p70 immunodepletion. The protein concentration of the MIA-PaCa-2 and OVCAR-8 cell extracts was 2.5 and 15 mg/ml, respectively. Native β-actin band intensity was recoded using a PhosphorImager. The amount of native β-actin measured after immunodepletion is expressed as a fraction of the amount measured for the cell extracts before immunodepletion in (B) MIA-PaCa-2 and (C) OVCAR-8 cell extracts.