Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: Only enzymatically active FKBP38 interacts with Bcl-2. (A) Co-immunoprecipitation of endogenous FKBP38 and Bcl-2. After preincubation with 500 μM EGTA, SH-SY5Y cell lysate was incubated with rabbit anti-FKBP38 antibody. Antibody/protein complexes were bound to protein G-Sepharose. Samples were washed. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using mouse anti-Bcl-2 antibody. Cell lysate preincubated with rabbit immunoglobin was used as a control. FKBP38 bound to Bcl-2 in the presence of 1 mM calcium. This interaction was disrupted by 1 μM GPI1046. No interaction was observed in the absence of Ca2+. (B) SH-SY5Y crude cell extract was applied in the absence and presence of 20 μM CaM and 2 mM Ca2+ to maltose-binding protein-Bcl-2 fusion protein (MBP-Bcl-2) immobilized on amylose resin. After three washing steps, protein was eluted by 200 mM maltose and analyzed by Western blot using polyclonal anti-FKBP38 antibody. The proportion of active endogenous FKBP38 was quantified by Biorad Multi-Analyst software. (C) MBP-Bcl-2 was immobilized on amylose resin and incubated with (1) FKBP38, (2) FKBP38 and CaM, (3) FKBP38 and Ca2+/CaM and (4) FKBP38, Ca2+/CaM and 200 nM GPI1046. After washing, protein was eluted and analyzed by Western blot using polyclonal anti-FKBP38 antibody. (D) Inhibition of PPIase activity of 1 μM FKBP38 by Bcl-2 was measured in the PPIase assay in the presence of 5 mM CaCl2 and 5 μM CaM. The calculated Ki value was 0.74 μM.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot, Binding Assay, Software, Inhibition, Activity Assay

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: FKBP38 activity influences Bcl-2 function. (A) Near-UV CD spectra of 1 μM Ca2+/CaM/FKBP38 and 3 μM Bcl-2 were measured either separated (dotted line) or mixed (solid line) in a tandem cuvette. (B) Co-immunoprecipitation. SH-SY5Y cells were stimulated to apoptosis by 50 μM etoposide. Cell lysate preincubated with 500 μM EGTA was incubated with monoclonal mouse anti-Bcl-2 antibody. Antibody/protein complexes were bound to protein G-Sepharose. Samples were washed. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using rabbit anti-FKBP38 antibody. Cell lysate preincubated with control mouse immunoglobin was used as a control. FKBP38 bound to Bcl-2 in the presence of 1 mM Ca2+, whereas 1 μM GPI1046 abolished the interaction. Bcl-2/Bad interaction was investigated by incubation of SH-SY5Y cell lysate with hamster anti-Bcl-2 antibody and protein was detected using mouse anti-Bad antibody. Bad/Bcl-2 interaction was observed in the presence of 1 mM calcium and 1 μM GPI1046. In addition, SH-SY5Y cell lysate was incubated with monoclonal mouse anti-Bad antibody, and antibody/protein complexes formed were bound to protein G-Sepharose. Precipitates (lanes 1–3) and input were subjected to SDS–PAGE and analyzed by Western blot using hamster anti-Bcl-2 antibody. Cell lysate preincubated with mouse immunoglobin was used as a control. Bcl-2 bound to Bad in the presence of 1 mM Ca2+ and 1 μM GPI1046, indicating that GPI1046 interfered with the Bcl-2/FKBP38 interaction, allowing Bad/Bcl-2 complexes to form. (C) MBP-Bcl-2 fusion protein was immobilized on amylose beads and incubated with Bad (Sigma), FKBP38 and CaM either in the presence or absence of 2 mM Ca2+. Pellet (P) and supernatant (S) were subjected to 12.5% SDS–PAGE and analyzed by Western blotting using mouse anti-Bad antibody and hamster anti-Bcl-2 antibody. (D) Subcellular distribution of Bcl-2 and Bad in SH-SY5Y neuroblastoma cells was analyzed by immunostaining with FITC-conjugated goat anti-mouse antibody against mouse anti-Bad antibody and Cy5-conjugated goat anti-hamster antibody against hamster anti-Bcl-2 antibody. Nuclei were stained with DAPI. Cells were treated for 16 h with 50 μM etoposide either in the absence or presence of 1 μM GPI1046. Cells transfected with FKBP38 RNAi construct were analyzed with ImageBlue-conjugated goat anti-mouse antibody against mouse anti-Bad antibody and TRITC-conjugated goat anti-hamster antibody against hamster anti-Bcl-2. Nuclei were stained by 7-AAD. Because of better understanding, colors of this panel were adapted to other results presented in this figure. Again, inhibition of FKBP38 activity allows Bcl-2/Bad interaction after induction of apoptosis by 50 μM etoposide.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Activity Assay, Immunoprecipitation, Incubation, SDS Page, Western Blot, Immunostaining, Staining, Transfection, Construct, Inhibition

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: FKBP38 and Bcl-2 colocalize in neuroblastoma cells. (A) Localization of Bcl-2 and FKBP38 in SH-SY5Y neuroblastoma cells was analyzed by immunostaining with Cy5-conjugated goat anti-hamster antibody against hamster anti-Bcl-2 antibody and FITC-conjugated goat anti-rabbit IgG against rabbit anti-FKBP38 antibody. Cells were treated for 16 h with 50 μM etoposide and 2 μM GPI1046. Nuclei were stained with DAPI. (B) Subcellular distribution of FKBP38 in SH-SY5Y cells was studied by immunostaining with FITC-conjugated goat anti-rabbit IgG against rabbit anti-FKBP38 antibody and a subcellular structure localization kit (Chemicon).

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Immunostaining, Staining

Journal:

Article Title: Bcl-2 regulator FKBP38 is activated by Ca 2+ /calmodulin

doi: 10.1038/sj.emboj.7600739

Figure Lengend Snippet: (C) Subcellular localization of FKBP38 and Bcl-2 was studied by preparing mitochondria, ER, nucleus and cytosol from SH-SY5Y cells and analyzing these fractions by Western blotting using anti-FKBP38 and anti-Bcl-2 antibodies. Antibodies detecting cytochrome c (Cyt c) and FKBP13 were used as controls for mitochondrial and ER localization, respectively.

Article Snippet: Bcl-2-binding assay : A 40 μl volume of 6 μM MBP-Bcl-2 fusion protein (MBP-Bcl-2) was subjected to 40 μl amylose resin (New England Biolabs, Beverly, MA) and incubated for 30 min.

Techniques: Western Blot

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 1. Expression levels of huOB-Ra mRNA in HTB-26 (A) and ZR75-1 (B) cells over time in response to treatment with human recombinant leptin. Data are representative of the average of two independent experiments. All ratios were determined by dividing the huOB-Ra cDNA band density value by its respective 18S cDNA band density value, and normalizing to the 0 h, 0 ng/ml leptin data point. *P<0.05, treated vs. control.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: Expressing, Recombinant, Control

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 2. Expression levels of huOB-Rb mRNA in HTB-26 (A) and ZR75-1 (B) cells over time in response to treatment with human recombinant leptin. Data are representative of the average of two independent experiments. All ratios were determined by dividing the huOB-Rb cDNA band density value by its respective 18S cDNA band density value, and normalizing to the 0 h, 0 ng/ml leptin data point.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: Expressing, Recombinant

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 3. Expression levels of SOCS-3 mRNA in HTB-26 (A) and ZR75-1 (B) cells over time in response to treatment with human recombinant leptin. Data are representative of the average of two independent experiments. All ratios were determined by dividing the SOCS-3 cDNA band density value by its respective 18S cDNA band density value, and normalizing to the 0 h, 0 ng/ml leptin data point. *P<0.01, treated versus control.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: Expressing, Recombinant, Control

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 4. Leptin-activated PI3K and MAPK signal transduction pathways in HTB-26 breast cancer cells. Cells were treated with 0, 4, 40, or 80 ng/ml of leptin for 0, 0.5, 1, 2, or 4 h. Representative Western blots are shown. Activation of the PI3K pathway was verified via phosphorylation of AKT (p-Akt) with cells treated with different concentrations of leptin (A, 4-h time point shown) over different periods of time (B, 40 ng/ml treatment shown). Likewise, activation of the MAPK pathway was verified via phosphorylation of ERK (p-ERK) with cells treated with different concentrations of leptin (C, 4-h time point shown) over different periods of time (D, 40 ng/ml treatment shown). Densitometry analysis was normalized to the total AKT or total ERK in each cell lysate.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: Transduction, Western Blot, Activation Assay, Phospho-proteomics

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 5. Leptin activates MAPK signal transduction pathways in ZR75-1 breast cancer cells. Cells were treated with 0, 4, 40, or 80 ng/ml of leptin for 0, 0.5, 1, 2, or 4 h. Representative Western blots are shown. Activation of the MAPK pathway was verified via phosphorylation of ERK (p-ERK) with cells treated with different concentrations of leptin (A, 4-h time point shown) over different periods of time (B, 40 ng/ml treatment shown). Densitometry analysis was normalized to the total ERK in each cell lysate.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: Transduction, Western Blot, Activation Assay, Phospho-proteomics

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 7. Effects of MAPK inhibitor U0126 and PI3K inhibitor LY294002 (LY) on leptin-induced proliferation in ZR75-1 breast cancer cells. Quiescent cells were exposed to 10% FBS in the presence or absence of 40 ng/ml leptin, with or without LY294002 or U0126. Cell proliferation was determined using an MTT assay and data was normalized to the 10% FBS control. (A) Cell proliferation was evaluated at 24, 48 and 72 h post treatment: 10% FBS (---), leptin (x), 40 μM LY294002 (•), leptin + 40 μM LY294002 (◊), 20 μM U0126 (▲), leptin + 20 μM U0126 (Δ). (B) ZR75-1 cell proliferation 48 h post leptin treatment.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: MTT Assay, Control

Journal: International Journal of Oncology

Article Title: Leptin receptor expression and cell signaling in breast cancer

doi: 10.3892/ijo.28.4.985

Figure Lengend Snippet: Figure 6. Effects of MAPK inhibitor U0126 and PI3K inhibitor LY294002 (LY) on leptin-induced proliferation in HTB-26 breast cancer cells. Quiescent cells were exposed to 10% FBS in the presence or absence of 40 ng/ml leptin, with or without LY294002 or U0126. Cell proliferation was determined using an MTT assay and data was normalized to the 10% FBS control. (A) Cell proliferation was evaluated at 24, 48 and 72 h post treatment: 10% FBS (---), leptin (x), 40 μM LY294002 (•), leptin + 40 μM LY294002 (◊), 20 μM U0126 (▲), leptin + 20 μM U0126 (Δ). (B) HTB-26 cell proliferation 48 h post leptin treatment.

Article Snippet: Cells were then serum-deprived for 20 h and then treated with 0, 4, 40 or 80 ng/ml of human recombinant leptin (R&D Systems, Minneapolis, MN) for 0, 0.5, 1, 2, 4, 24 or 48 h. Two independent tissue culture experiments were analyzed per cell line.

Techniques: MTT Assay, Control