anti inositol 1 4 5 trisphosphate type 2 receptor  (Santa Cruz Biotechnology)

Journal: Cell death and differentiation

Article Title: The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1.

doi: 10.1038/cdd.2009.34

Figure Lengend Snippet: Figure 5 Xestospongin B effects are mTOR-independent. HeLa cells were treated with xestospongin B (2 mM) or rapamycin (1 mM) for the indicated periods and the levels of mTOR and p70S6K phosphorylation were assessed by immunoblotting (a). The Beclin 1/IP3R interaction was not modified by rapamycin treatment, in conditions in which xestospongin B caused its dissociation (b). Results are presented as mean±S.E.M. of three independent triplicate assessments. **Po0.01 versus untreated controls

Article Snippet: Primary antibodies specific for Bcl-2, Beclin 1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), IP3R-I (Calbiochem), IP3R types I-II-III (Santa Cruz), LC3B, mTOR, p70S6K, phospho-mTOR or phospho-p70S6K (Cell Signaling, Danvers, MA, USA) were incubated overnight at 4 1C and revealed with the appropriate horseradish peroxidase-labeled secondary antibodies (SouthernBiotech, Birmingham, AL, USA) by means of the SuperSignal West Pico chemoluminiscent substrate (Pierce, Rockford, IL, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: Cell death and differentiation

Article Title: The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1.

doi: 10.1038/cdd.2009.34

Figure Lengend Snippet: Figure 7 Overexpression of the IP3R-LBD inhibits xestospongin B-induced autophagy through interaction with Beclin 1. HeLa cells were cotransfected with GFP-LC3 and three IP3R-LBD-RFP chimeric proteins targeted to different subcellular compartments, namely the cytosol (Cyto), the outer mitochondrial membrane (OMM) and the endoplasmic reticulum (ER). An inactive ER-targeted IP3R-LBD-RFP, including a non-apeptide linker (9aaER) as well as equally targeted variants of RFP alone were used as controls. Twenty-four hours after transfection, cells were subjected to treatment with xestospongin B or nutrient starvation. Representative pictures of Hoechst 33342- counterstained cells were taken 4 h after treatment (a) and the percentage of adherent cells exhibiting GFP-LC3 vacuolization into cytoplasmic puncta was determined (b). The interaction between the IP3R-LBD and Beclin 1 was assessed by immunoprecipitation experiments in HeLa cells that were transiently expressing IP3R-LBD-ER-RFP and concomitantly subjected to siRNA-mediated emerin or Bcl-2 knockdown as indicated (c). Coimmunoprecipitated Beclin 1 levels were normalized relative to input protein levels (d). LC3-II levels from the input lysates were normalized relative to GAPDH levels (e). Data are reported as mean±S.E.M. of three independent experiments carried out in triplicates. *Po0.05 and **Po0.01. The colour reproduction of this figure is available on the html full version of the manuscript

Article Snippet: Primary antibodies specific for Bcl-2, Beclin 1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), IP3R-I (Calbiochem), IP3R types I-II-III (Santa Cruz), LC3B, mTOR, p70S6K, phospho-mTOR or phospho-p70S6K (Cell Signaling, Danvers, MA, USA) were incubated overnight at 4 1C and revealed with the appropriate horseradish peroxidase-labeled secondary antibodies (SouthernBiotech, Birmingham, AL, USA) by means of the SuperSignal West Pico chemoluminiscent substrate (Pierce, Rockford, IL, USA).

Techniques: Over Expression, Membrane, Transfection, Immunoprecipitation, Expressing, Knockdown

Journal: Biochimica et biophysica acta

Article Title: Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets.

doi: 10.1016/j.bbamcr.2007.12.008

Figure Lengend Snippet: Fig. 3. Microtubules are involved in the coupling between hTRPC1 and the type II IP3R stimulated by selective depletion of the DTS or the acidic stores in human platelets. Human platelets were treated with 20 µM TBHQ (A), 10 nM TG (B) or the vehicles, as controls (Non-stimulated) and then lysed. Whole-cell lysates were immunoprecipitated (IP) with anti- IP3RII antibody. Immunopre- cipitates were analysed by Western blotting (WB) using anti-hTRPC1 antibody (top panel) or anti-IP3RII antibody (bottom panel) as described in the Materials and methods section. Positions of molecular-mass markers are shown on the right. These results are representative of 4–7 independent experiments.

Article Snippet: Anti-IP3R type II polyclonal antibody and horseradish-peroxidaseconjugated donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Techniques: Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets.

doi: 10.1016/j.bbamcr.2007.12.008

Figure Lengend Snippet: Fig. 4. Colchicine impairs coupling between hTRPC6 and the type II IP3R stimulated by selective depletion of the DTS or the acidic stores in human platelets. Human platelets were preincubated for 30 min in the presence of 30 µM colchicine (Colchicine) or the vehicle (Control). Cells were then treated with 20 µM TBHQ (A), 10 nM TG (B) for several times (30 s–3 min) or the vehicles, as controls (NS) and then lysed. Whole-cell lysates were immunoprecipitated (IP) with anti-IP3RII antibody. Immunoprecipitates were analysed by Western blotting (WB) using anti-hTRPC6 antibody as described in the Materials and methods section. Positions of molecular-mass markers are shown on the right. Results are expressed as percentage of control (resting cells not treated with colchicine) and are presented as means±S. E.M. of 4–5 separate experiments.

Article Snippet: Anti-IP3R type II polyclonal antibody and horseradish-peroxidaseconjugated donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Techniques: Control, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets.

doi: 10.1016/j.bbamcr.2007.12.008

Figure Lengend Snippet: Fig. 6. Colchicine impairs the interaction of SERCA3 with hTRPC1 and 6 and the type II IP3 receptor stimulated by selective depletion of the acidic stores in human platelets. Human platelets were preincubated for 30 min in the presence of 30 µM colchicine (Colchicine) or the vehicle (Control). Cells were then treated with 20 µM TBHQ for several time periods (30 s–3 min) or the vehicles, as controls (NS) and then lysed. Whole-cell lysates were immunoprecipitated (IP) with anti-SERCA3 antibody (A and B) or anti-IP3RII antibody (C). Immunoprecipitates were analysed by Western blotting (WB) using anti-hTRPC1 (A), anti-hTRPC6 (B) or anti- SERCA3 (C) antibodies as described in the Materials and methods section. Positions of molecular-mass markers are shown on the right. Results are expressed as percentage of control (resting cells not treated with colchicine) and are presented as means±S.E.M. of 4–7 separate experiments.

Article Snippet: Anti-IP3R type II polyclonal antibody and horseradish-peroxidaseconjugated donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Techniques: Control, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets.

doi: 10.1016/j.bbamcr.2007.12.008

Figure Lengend Snippet: Fig. 7. Electrotransjection of anti-SERCA3 antibody (PL/IM430) attenuates the interaction of the type II IP3 receptor with hTRPC1 and hTRPC6. Human platelets (109 cells/mL) were electropermeabilized in a Gene Pulser as descri- bed in Material and methods. Following electroporation, cells were incubated in the absence (−) or presence of 2 µg/mL anti-SERCA3 (PL/IM430) antibody (α-SERCA) or mouse anti-IgG (m-IgG), as indicated, for an additional 60 min at 37 °C, centrifuged at 350 ×g for 20 min and resuspended in HBS containing 200 µM CaCl2. At the time of experiment 250 µM EGTA was added. Human platelets were treated with 20 µM TBHQ or the vehicle, as control, and then lysed. Whole-cell lysates were immunoprecipitated (IP) with anti- IP3RII an- tibody. Immunoprecipitates were analysed by Western blotting (WB) using anti- hTRPC1 antibody (A, top panel), anti-hTRPC6 antibody (B, top panel) or anti- IP3RII antibody (A and B, bottom panels) as described in the Materials and methods section. Positions of molecular-mass markers are shown on the right. These results are representative of 6 independent experiments.

Article Snippet: Anti-IP3R type II polyclonal antibody and horseradish-peroxidaseconjugated donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Techniques: Electroporation, Incubation, Control, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets.

doi: 10.1016/j.bbamcr.2007.12.008

Figure Lengend Snippet: Fig. 9. Colchicine impairs the interaction of SERCA2b with hTRPC1 and 6 and the type II IP3 receptor stimulated by selective depletion of the DTS in human platelets. Human platelets were preincubated for 30 min in the presence of 30 µM colchicine (Colchicine) or the vehicle (Control). Cells were then treated with 10 nM TG for several time periods (1–3 min) or the vehicle, as controls (NS) and then lysed. Whole-cell lysates were immunoprecipitated (IP) with anti-SERCA2b antibody (A and B) or anti-IP3RII antibody (C). Immunoprecipitates were analysed by Western blotting (WB) using anti-hTRPC1 (A), anti-hTRPC6 (B) or anti- SERCA2b (C) antibodies as described in the Materials and methods section. Positions of molecular-mass markers are shown on the right. Results are expressed as percentage of control (resting cells not treated with colchicine) and are presented as means±S.E.M. of 6 separate experiments.

Article Snippet: Anti-IP3R type II polyclonal antibody and horseradish-peroxidaseconjugated donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Techniques: Control, Immunoprecipitation, Western Blot