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. 2012 Aug 9;3(8):e367.
doi: 10.1038/cddis.2012.103.

Bax inhibitor-1 Is a Novel IP₃ Receptor-Interacting and -Sensitizing Protein

Free PMC article

Bax inhibitor-1 Is a Novel IP₃ Receptor-Interacting and -Sensitizing Protein

S Kiviluoto et al. Cell Death Dis. .
Free PMC article


Figure 1
Figure 1
(a) Interaction of 5xMyc-BI-1 and 5xMyc-BI-1D213R with IP3R channels. BI-1 and BI-1D213R were expressed as 5xMyc-tagged fusion proteins. The empty 5xMyc vector was used as negative control. The vectors were transfected into HeLa cells for 2 days allowing the expression of the 5xMyc-tagged proteins. Cell lysates were prepared using 1% CHAPS buffer and the overexpressed 5xMyc-tagged proteins were purified using anti-Myc-agarose beads (Sigma, Saint Louis, MO, USA). After washing the beads three times with CHAPS buffer, proteins were eluted using urea sample buffer and the immunoprecipitated samples were analyzed via SDS-PAGE and western blotting analysis (antibodies used for immunoblotting are indicated above WB). The double line on the western blot indicates that lanes from another part of the same gel and exposure time were merged. Using this immunoprecipitation approach, we found that both IP3R1 and IP3R3 co-immunoprecipitated with 5xMyc-BI-1 and 5xMyc-BI-1D213R, but not with 5xMyc vector. (b) Residues 198–217 of human BI-1 were synthesized as a Flag-tagged peptide (Flag-CTP1), which was applied in co-immunoprecipitation experiments using anti-Flag-M2-agarose beads (Sigma) and cell lysates from DT40 triple-IP3R knockout cells ectopically expressing IP3R1. The double line on the western blot indicates that lanes from another part of the same gel and same exposure time were merged. From the western blot analysis using the Rbt03 anti-IP3R1 C-terminal antibody, it is clear that full-length IP3R1 as well as C-terminal fragments interacted with Flag-CTP1 (indicated as IP3R1-Cterm). The numbers indicate Mw markers in kilodalton. (c) Inset is an RT-PCR showing similar mRNA-expression levels of ectopically expressed BI-1 and BI-1D213R in BI-1−/− MEF cells using a bi-cistronic C-terminal IRES-RFP reporter as vector. The main panel shows the results obtained from unidirectional 45Ca2+-flux assays in saponin-permeabilized BI-1−/− MEF cells comparing IP3-induced Ca2+ release between vector-expressing, BI-1-expressing and BI-1D213R-expressing cells, indicating IP3R sensitization by BI-1 independent of BI-1's Ca2+-flux properties. For analysis, cells were grown to the same density to perform an accurate comparison of the IP3-induced Ca2+-release responses between the different cell lines. Data represent mean±S.E.M. from three to five independent experiments using two replicates

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