JCS03001 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Diagram of hTERT protein indicating the regions of hTERT used as immunogens to develop anti-hTERT antibodies. A scale diagram indicating functionally important regions of hTERT (Armbruster et al., 2001; Beattie et al., 2000; Lai et al., 2001; Moriarty et al., 2002; Nakamura et al., 1997; Xia et al., 2000). The enzyme contains seven motifs characteristic of reverse transcriptases and an RNA-binding region composed of conserved domains II (CP), III (QFP), and the T motif. Additionally, the enzyme contains two conserved N-terminal (I-II) and four C-terminal (I-IV) domains necessary for catalytic activity. There are two biologically important DAT domains that ‘Dissociate Activities of Telomerase’, where mutations left the enzyme catalytically active, but unable to function in vivo. Numbers indicate amino acid positions in the human telomerase sequence. The immunological properties of the various antibodies are shown in Table 1.

• Supplemental Figure 2 -

  Fig. S2. hTERT mRNA expression in the various cell lines used in the study. hTERT mRNA expression was quantified by fluorescence real-time RT-PCR using the LightCycler® technology and the LightCycler TeloTAGGG hTERT Kit from according to the manufacturer’s (Roche Diagnostics) instructions. The hTERT level was normalized to the expression of the housekeeping gene porphobilinogen deaminase (PBGD).

• Supplemental Figure 3 -

  Fig. S3. Detection of HA-tagged hTERT in HeLa cells. Western blot analyses of total cell lysates from HeLa cells transfected with either HA-tagged hTERT (pB-hTERT-HA), the non-tagged hTERT protein (pB-hTERT), or p-Babe empty vector alone (pB) using anti-HA antibody.

• Supplemental Figure 4 -

  Fig. S4. Immunoblot analysis of NB4-LR1/hTERT immunoprecipitates. The indicated antibodies were used to immunoprecipitate proteins from NB4-LR1/hTERT cells. The immunoprecipitates were resolved by SDS-PAGE and western blotted with anti-Hsp90b and nucleolin (A), and anti-alpha-actinin (B) antibodies. Positions of the molecular markers are indicated on the left. Positions of the proteins of interest are indicated by arrows. Position of the heavy chain (Ig) of antibodies is shown. IgG, control for nonspecific binding by performing immunoprecipitation reaction using normal mouse or rabbit IgG. Note that alpha-actinin is found in NCL-hTERT but not in RCK-hTERT immunoprecipitates (B, lane 1 and 3).