Journal: Journal of Histochemistry and Cytochemistry

Article Title: Euptox A Induces G1 Arrest and Autophagy via p38 MAPK- and PI3K/Akt/mTOR-Mediated Pathways in Mouse Splenocytes

doi: 10.1369/0022155417722118

Figure Lengend Snippet: Antibodies Used in the Study.

Article Snippet: Bcl-2 , Rabbit, polyclonal , D17C4 , Cell Signaling Technology.

Techniques:

Journal: Leukemia

Article Title: BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells.

doi: 10.1038/leu.2008.175

Figure Lengend Snippet: Figure 2 Protein expression of antiapoptotic BCL-2 family proteins in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) primary cells. (a) Total protein extracts (50 mg) from representative CLL and MCL samples were analyzed by western blot for MCL-1, BCL-XL, BCL- 2, pBCL-2(Ser70) and a-tubulin expression. (b) Relative protein quantification of MCL-1, BCL-XL, BCL-2 and pBCL-2(Ser70) was performed using Image Gauge Fujifilm software and a-tubulin levels for loading normalization. The pool of BCL-2 protein levels (left), calculated as the sum of MCL-1, BCL-2 and BCL-XL levels, and pBCL-2/BCL-2 ratio (right), are shown for each CLL (n ¼ 20; ’) and MCL (n ¼ 8; m) patient. (c) pBCL-2/BCL- 2 ratios were compared in cells from CLL patients with high (n ¼ 8; ’) or low (n ¼ 12; &) ZAP-70 expression. Mean expression value for each group is represented with a horizontal line. In all analysis, protein extract from CLL patient no. 14 was the internal control used to normalize samples analyzed in different gels.

Article Snippet: The following monoclonal and polyclonal antibodies were used: MCL-1 (S-19), BCL-XL/S (2H12), phospho-ERK1/2 and BCL-2 (100; Santa Cruz Biotechnology, Santa Cruz, CA, USA); MCL-1 (clone 22; BD-Pharmingen, San Diego, CA, USA); BCL-2 (Dako, Glostrup, Denmark); BIM (Calbiochem); BAK (NT) (Upstate, Lake Placid, NY, USA); phospho-BCL-2 (Ser70) (Cell Signalling Technologies, Beverly, MA, USA) and anti-a-tubulin (Sigma).

Techniques: Expressing, Western Blot, Software, Control

Journal: Leukemia

Article Title: BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells.

doi: 10.1038/leu.2008.175

Figure Lengend Snippet: Figure 3 BCL-2 phosphorylation modulates sensitivity to GX15-070 and is regulated by extracellular signal-regulated kinase (ERK) and PP2A phosphatase activities in chronic lymphocytic leukemia (CLL) cells. (a) Parental H1299 cells and H1299-expressing BCL-2b5 or S70E-BCL-2b5 were treated with 0.5 mM GX15-070 for 48 h and Annexin V-positive cells were analyzed. Western blot analysis of BCL-2 and a-tubulin for each cell line is also shown. (b) Correlation between the expression levels of pERK and pBCL-2/BCL-2 ratios in CLL samples from Figure 2a. (c) Cells from four representative CLL patients were treated for 6 h with 20 mM PD98059 and total protein extracts were analyzed by western blot for the expression of pERK, pBCL-2(Ser70), BCL-2 and a-tubulin. pBCL-2/BCL-2 ratios calculated using Image Gauge Fujifilm software. (d) Cells from four representative CLL patients were cotreated with 20 mM PD98059 and 1 mM GX15-070 for 40 h and viability was assessed by DiOC6[3] labeling and fluorescence-activated cell sorting (FACS) analysis. Percentage of response calculated refers to each patient’s control. Combination indexes (CIs) were calculated using Calcusyn software. (e) Cells from two representative CLL patients were treated with 1 nM okadaic acid (OA) for the times indicated and total protein extracts were analyzed for pBCL-2, BCL-2 and a-tubulin expression. (f) Cells from four representative CLL patients were cotreated with 1 nM OA and 1 mM GX15-070 for 40 h and viability was assessed by DiOC6[3] labeling. Percentage of response was calculated referred to each patient’s control.

Article Snippet: The following monoclonal and polyclonal antibodies were used: MCL-1 (S-19), BCL-XL/S (2H12), phospho-ERK1/2 and BCL-2 (100; Santa Cruz Biotechnology, Santa Cruz, CA, USA); MCL-1 (clone 22; BD-Pharmingen, San Diego, CA, USA); BCL-2 (Dako, Glostrup, Denmark); BIM (Calbiochem); BAK (NT) (Upstate, Lake Placid, NY, USA); phospho-BCL-2 (Ser70) (Cell Signalling Technologies, Beverly, MA, USA) and anti-a-tubulin (Sigma).

Techniques: Phospho-proteomics, Expressing, Western Blot, Software, Labeling, FACS, Control

Journal: Leukemia

Article Title: BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells.

doi: 10.1038/leu.2008.175

Figure Lengend Snippet: Figure 4 Effect of BCL-2 phosphorylation on GX15-070 plus bortezomib combination. (a) Chronic lymphocytic leukemia (CLL) cells were preincubated with GX15-070 (0.1, 0.5, 1 and 2 mM) for 20 h and then 5 nM bortezomib was added for 20 additional hours. Combination indexes (CIs) were calculated for each combination and plotted vs pBCL-2/BCL-2 ratio for each CLL sample. Linear regression and statistical significance were defined using GraphPad Prism software. (b) Cells from CLL patient no. 2 were treated with 10 nM bortezomib for the times indicated. Total protein extracts were analyzed by western blot for BCL-2, pBCL-2(Ser70) and a-tubulin expression, and pBCL-2/BCL-2 ratios were calculated. (c) Cells from CLL patient no. 3 were preincubated for 1 h with 20 mM PD98059, and then treated with 10 nM bortezomib for 5 additional hours. Total proteins extracts were analyzed for the expression of pERK, pBCL-2(Ser70), BCL-2 and a-tubulin, and pBCL-2/BCL-2 ratios calculated as above. (d) Cells from two representative CLL patients were treated with the previously indicated doses of GX15-070 alone for 40 h, together with either 5 nM bortezomib or with both 5 nM bortezomib plus 20 mM PD98059. Viability was assessed by DiOC6[3] labeling and fluorescence-activated cell sorting (FACS) analysis and percentage of response calculated referred to each patient control. CIs calculated as above, are indicated inside the plot for each combination.

Article Snippet: The following monoclonal and polyclonal antibodies were used: MCL-1 (S-19), BCL-XL/S (2H12), phospho-ERK1/2 and BCL-2 (100; Santa Cruz Biotechnology, Santa Cruz, CA, USA); MCL-1 (clone 22; BD-Pharmingen, San Diego, CA, USA); BCL-2 (Dako, Glostrup, Denmark); BIM (Calbiochem); BAK (NT) (Upstate, Lake Placid, NY, USA); phospho-BCL-2 (Ser70) (Cell Signalling Technologies, Beverly, MA, USA) and anti-a-tubulin (Sigma).

Techniques: Phospho-proteomics, Software, Western Blot, Expressing, Labeling, FACS, Control