Journal:

Article Title: Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

doi: 10.1093/emboj/19.22.5971

Figure Lengend Snippet: Fig. 1. Electron microscopy of CCT–β-tubulin complexes. (A) A gallery of top views obtained by negative staining (NS) or cryoelectron microscopy (CR). (B) Two-dimensional average of CCT–β-tubulin shown as contour lines (average of 2313 particles). (C) A view of three-dimensional reconstructions of the CCT–β-tubulin complexes obtained from stained specimens. Approximately 40 Å of the height of the CCT particle is reconstructed.

Article Snippet: Either β-tubulin or bovine brain tubulin (from Cytoskeleton) were denatured in 7 M guanidinium chloride and incubated in diluting buffer (100-fold) containing 0.4 µM murine CCT purified as described by Liou and Willison (1997) .

Techniques: Electron Microscopy, Negative Staining, Microscopy, Staining

Journal:

Article Title: Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

doi: 10.1093/emboj/19.22.5971

Figure Lengend Snippet: Fig. 4. Docking of the atomic structures of actin and tubulin. (A) Atomic structures of actin (Kabsch et al., 1990) and tubulin (Nogales et al., 1998a). Both proteins are divided into two domains: the N-terminal domain (red domain) and the C-terminal domain (yellow and white domains in actin and yellow, green and white domains in tubulin). The blue region corresponds to the nucleotide (ADP in actin, GTP in tubulin). (B) Docking of the two domains of the atomic structure of actin with the cryo-electron microscopy three-dimensional reconstruction of actin complexed to CCT (Llorca et al., 1999b). The code colours are the same as in (A), with the white domain corresponding to the fragment that permits a chimeric protein to bind strongly to CCT (β-actin.sub4) (Llorca et al., 1999b). (C) Docking of the two domains of the atomic structure of tubulin with the cryo-electron microscopy three-dimensional reconstruction of tubulin complexed to CCT (Figure 2). The code colours are the same as in (A), with the white, green and yellow domains corresponding to the tubulin fragments inserted in the chimeric proteins Ha-Ras–β-tubulin fragments 5, 3 and 4, respectively. (D) Enlarged side view of the docking of actin shown in (B). (E) Enlarged side view of the docking of tubulin shown in (C).

Article Snippet: Either β-tubulin or bovine brain tubulin (from Cytoskeleton) were denatured in 7 M guanidinium chloride and incubated in diluting buffer (100-fold) containing 0.4 µM murine CCT purified as described by Liou and Willison (1997) .

Techniques: Electron Microscopy

Journal:

Article Title: Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

doi: 10.1093/emboj/19.22.5971

Figure Lengend Snippet: Fig. 5. Two-dimensional average images of immunocomplexes of CCT–tubulin and CCT–chimeric proteins. (A and B) Average images of CCT–tubulin–8g (anti-CCTδ) complexes (average of 527 and 491 particles, respectively). (C and D) Average images of CCT–Ha-Ras–β-tubulin fragment 5–PK-5h (anti-CCTθ) complexes (average of 345 and 381 particles, respectively). (E and F) Average images of CCT–Ha-Ras–β-tubulin fragment 3–8g (anti-CCTδ) complexes (average of 293 and 122 particles, respectively). (G and H) Average images of CCT–Ha-Ras–β-tubulin fragment 4–8g (anti-CCTδ) complexes (average of 208 and 220 particles, respectively). (I) A model depicting the two possible modes of tubulin interaction with CCT, using the CCT subunit orientation published by Liou and Willison (1997), which is fully consistent with the results obtained here. The colour codes are as in Figure 4A and C. All the average images shown in (A)–(H) are oriented as in (I).

Article Snippet: Either β-tubulin or bovine brain tubulin (from Cytoskeleton) were denatured in 7 M guanidinium chloride and incubated in diluting buffer (100-fold) containing 0.4 µM murine CCT purified as described by Liou and Willison (1997) .

Techniques:

Journal:

Article Title: Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

doi: 10.1093/emboj/19.22.5971

Figure Lengend Snippet: Fig. 6. Interaction of α-tubulin, β-tubulin and fragments of β-tubulin with CCT following in vitro translation. (A) Native PAGE analysis of in vitro translation at 30 min time points of full-length β-tubulin (wt), Ha-Ras–β-tubulin fragment 1 (f1; red colouring in Figure 4C), Ha-Ras–β-tubulin fragment 2, a combination of fragments 3 and 5 (f2), Ha-Ras–β-tubulin fragment 3 (f3; green in Figure 4C), Ha-Ras–β-tubulin fragment 5 (f5, yellow in Figure 4C) and Ha-Ras–β-tubulin fragment 4 (f4; white in Figure 4C). The sequence of the fragments is described in Materials and methods. (B) Quantitative analysis of an in vitro translation experiment in rabbit reticulocyte lysate over 10–120 min with full-length β-tubulin (squares) and Ha-Ras–β-tubulin fragment 5 (triangles). The degree of association of translated proteins with CCT was determined by calculating the ratio between the CCT counts and the total counts in the lane. (C) Autoradiograms showing recovery of [35S]α-tubulin and [35S]β-tubulin from the 20S sucrose peak by immunoprecipitation with a set of anti-CCT subunit antibodies in NP-40 (lane N) and mixed micelle buffers (lanes α to ζ-1), as described by Hynes and Willison (2000). Lane S indicates starting sample for immunoprecipitation (i.e. 20S sucrose fraction), lane N indicates recovery of intact 35S-labelled tubulin–CCT complexes in 0.5% NP-40 and lane B indicates background signal obtained by incubation of starting sample with beads alone in mixed micelle buffer. Samples displayed on 8% SDS–PAGE gel. Molecular mass markers (kDa) are indicated in the right hand margin of the autoradiogram. The lower species (asterisk) is an internally initiated β-tubulin polypeptide. (D and E) Quantitation of recovery of [35S]α-tubulin and [35S]β-tubulin, respectively, by each anti-CCT subunit antibody in mixed micelle buffer after subtraction of background signal (signals α to ζ-1). Lanes annotated as in (C).

Article Snippet: Either β-tubulin or bovine brain tubulin (from Cytoskeleton) were denatured in 7 M guanidinium chloride and incubated in diluting buffer (100-fold) containing 0.4 µM murine CCT purified as described by Liou and Willison (1997) .

Techniques: In Vitro, Clear Native PAGE, Sequencing, Immunoprecipitation, Incubation, SDS Page, Quantitation Assay

Journal: Journal of Veterinary Science

Article Title: In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

doi: 10.4142/jvs.2013.14.2.125

Figure Lengend Snippet: The effect of MEL on IL-10 (A), TGF-β (B), and IFN-γ (C) production. The results are expressed as a percentage of CD25 high CD4+, CD25 low CD4+, and CD25-CD4+ cells expressing IL-10, TGF-β, or IFN-γ. Typical cytograms (D) illustrating IFN-γ expression in particular lymphocyte subpopulations are shown. Results are presented as the mean ± SE (n = 18) of three independent experiments (each one using six animals). * p < 0.01, MEL-treated cells versus the control cells.

Article Snippet: After extracellular staining (as described above) and fixation (200 μL 2% paraformaldehyde in Dulbecco's PBS per sample for 15 min on ice; both reagents were purchased from Sigma-Aldrich, USA), the cells were permeabilized with 2 mL SAP buffer [0.1% (w/v) saponin and 0.05% (w/v) NaN 3 in Hanks' balanced salt solution (HBSS; all from Sigma-Aldrich, USA)] and stained with APC (allophycocyanin) -conjugated mouse anti-TGF-β mAb (1 : 20, 1D11, IgG1, this antibody reacts with human, mouse, and bovine TGF-β1 and TGF-β2; R&D Systems, USA) for 45 min at RT in the dark.

Techniques: Expressing, Control

Journal: Biology of Sex Differences

Article Title: Sex differences in disease severity and immune responses in murine and human inflammatory arthritis

doi: 10.1186/s13293-026-00840-w

Figure Lengend Snippet: Male CIA mice exhibit higher disease severity compared to females. Male and female CIA and saline control mice were monitored for disease severity and assigned clinical scores from day 21 after the first CII challenge until the end of experiment (day 29). (A) Line graphs representing the mean clinical scores of mice starting from day 1 to day 29. On day 29 after the first CII challenge, mice were euthanized by cardiac puncture under anesthesia for blood and serum collection and storage at −80 °C. Serum concentrations of (B) anti-mouse collagen type II antibodies (left panel) and anti-bovine collagen type II antibodies (right panel) were analyzed by ELISA. N = 5 mice per group. One of two independent experiments. Simple linear regression analysis was performed to determine the statistical difference between the lines. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test was used to determine the statistical significance between the groups. (*/ # p ≤ 0.05, ** p ≤ 0.005 and ***/ ### p ≤ 0.0005). In Fig. 1A, # significance of comparisons between CIA and saline control mice; *significance of comparisons between sexes of CIA mice

Article Snippet: Serum levels of mouse anti-collagen antibodies (autoantibodies) and bovine anti-collagen antibodies (antibodies to the immunizing antigen) were determined by ELISA using a Mouse Anti-mouse Type II Collagen IgG Antibody Assay Kit and Mouse Anti-Bovine Type II Collagen IgG Antibody Assay Kit, respectively, according to the manufacture’s protocol (Chondrex Inc. WA, USA).

Techniques: Saline, Control, Enzyme-linked Immunosorbent Assay, Comparison