Fig. 2. Histogram representing fold changes in the levels of mRNA determined by real time PCR, performed on mRNA extracted from postnatal developing mouse kidney. At postnatal (PN) day 1, 5, 10, 20, 30 and adult (24 weeks), the levels of mRNA corresponding to all three transcripts were quantified relative to the normalized value at PN 1. At PN 20, levels were significantly different from all other values (P < 0.001). In separate experiments, the relative levels of the three transcripts A (short), B (medium) and C (long) have been determined for PN 1, PN 20 and adult kidney.

Fig. 3. A representation of the secondary structure of the human CCT protein is shown and includes 12 transmembrane spans, each with a continuous sequence of approximately 20 amino acids each. These are largely lipophilic in nature and are thought to assume an α-helical configuration, a stiff structure that is considered long enough to transverse a membrane bilayer of polar phospholipids. The selection of probable transmembrane domains is based on analysis using the Kyte–Doolittle algorithm (data not shown). Epitopes 1 (KPNIEDRDELAYHIS, external loop 3) and 2 (KEKPKYPPSRAQS, internal loop 3) were used to design synthetic peptides for the production of antibodies.

Fig. 4. The alignment of sequences published on the databases from the N- and C-terminal domains and the interspanning (V–VI) domain from human, mouse, rat, pig (gi 14306042, 9017023) and cow (gi 11706376, 11917831) are compared to the C10 homologue of human. The N-terminal sequences have been spaced optimally to highlight the conserved theme of hexad repeats, and in the interspanning V–VI domain, the conservation of its canonical forms is shown (Sp = span).

Fig. 5. Immunofluorescence images of human pituitary gland. In this figure (a, c, e, g), cells expressing CCT are identified using the mouse monoclonal antibody 17/00. These are dual stained with rabbit polyclonal antibodies against (b) growth hormone to identify somatomammotrophs, (d) prolactin (lactotrophs), (f) ACTH (basophils) and (h) TSH (chromophobes). The horizontal bar in each represents 10 μm.

Fig. 6. Immunohistochemical staining in various tissues, in some cases with serial sections, and antibodies raised against two separate epitopes of CCT (anti-human epitope 1, PAb 10B; anti-rat epitope 1, PAb 9B; anti-human or rat epitope 2, MAb 33-01and PAb 126). Panels A (MAb 33-01, 1:100) and B (PAb 10B, 1:500) are serial sections of human postmortem pituitary. Arrows indicate matched positive cells and arrowheads indicate matched unstained cells. Basophil cells also do not stain using both antibodies. In panels C1 and C2 are images of tissue surrounding the same blood vessel and are negative controls for A and B in which the primary antibodies have been omitted. In panel D, a section of developing rat (E18) pituitary is shown in which individual cells that express CCT are stained using PAb 126 (1:200). In panel E, the surface of the follicular cells that line the ducts of the E18 developing rat thyroid is positive for CCT shown here using PAb 126 (1:200). Panels F–K represent sections of E21 bone. Serial sections F (TRAP stain) and G (PAb 189, anti-CTR, 1:250), serial sections I (MAb 33-01, 1:100) and J (PAb 9B, 1:1000), and further serial sections K1 (MAb 33-01, 1:200) and K2 (PAb 189, 1:500), together identify osteoclasts as expressing CCT protein. Negative controls of E21 bone stained in the absence of primary (H1 monoclonal and H2 polyclonal) antibodies. In panels L1 and L2 are shown chondrocytes of the developing cartilage (E19) in the inner ear (L1) and shoulder joint (L2) stained using MAb 33-01 (1:100). In L2-positive cells are aligned along the growth zones (arrows) in the region of the joint (arrowhead). In panels M1 and M2 are shown positive cardiomyocytes (E17), at lower power (M1) and at higher magnification (M2), stained using MAb 33-01 (1:200). In N1 (E17 fetal liver) and N2 (E19 fetal liver), positive cells include putative Kupfer cells lining the sinusoids (arrows) and haemopoeitic cells (arrowheads) have been identified using MAb 33-01 (1:200). Panels O (cortex) and P (medulla) illustrate structures of PN 30 mouse kidney, namely collecting ducts (arrowheads) and distal tubules (arrows) positively stained with MAb 33-01 (1:200). The negative controls for cortex and medulla are shown in Q1 and Q2. In the rat PN 30 kidney similar structures stained positively with MAb 33-01 (R1, 1:100) and PAb 9B (R2, 1:1000 in the renal cortex). The negative controls for these images correspond to those previously published [18]. In panels S (PAb 126, 1:1000) and T (negative control) are shown sections of mouse placenta in which staining identifies expression of CCT. In U1 and U2 are shown the results of staining with MAb 33/01 (1:200) in the retinal pigment epithelial cells adjacent to the choroid (detached retina in U1) and in vessels (U2) aligned along the inner layer of developing retina of an E18 rat fetus. Panels V1 and V2 show staining in cells from E17 fetal CNS lining the transition zone between the fourth ventricle and the central canal of the spinal cord stained using MAb 33-01 (1:200) and PAb 9B (1:1000), respectively. The horizontal bar shown in V2 represents 30 μm in D, L, M2, N, U and V, 50 μm in A–C, F–K and O–R, 75 μm in S and T and 100 μm in E and M1.