Journal: Cell Death and Differentiation

Article Title: A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis

doi: 10.1038/cdd.2016.102

Figure Lengend Snippet: BV6/Dexa-induced loss of MMP and cell death depend on Bak. (a) Cells were treated for 6 h with BV6 (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) and/or 200 μM Dexa, Jurkat cells were additionally treated with 20 μM zVAD.fmk. Bak activation was determined by IP using an active conformation-specific antibody. Protein expression of Bak was analyzed by western blotting. β-Actin served as loading control. (b) ALL cells were transiently transfected with two distinct siRNAs targeting Bak or control siRNA. Protein expression of Bak was analyzed by western blotting. β-Actin served as loading control. (c) Cells were treated for 24 h with BV6 (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) and/or 200 μM Dexa, Jurkat cells were additionally treated with 20 μM zVAD.fmk. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and S.D. of at least three independent experiments performed in triplicate are shown; *P<0.05. (d) Cells were treated for 6 h with BV6 (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) and 200 μM Dexa, Jurkat cells were additionally treated with 20 μM zVAD.fmk. Loss of MMP was determined by TMRM staining and flow cytometry. Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05. (e) Cells were treated for 4 h with BV6 (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) and/or 200 μM Dexa, Jurkat cells were additionally treated with 20 μM zVAD.fmk. ROS production was determined by CellROX staining and flow cytometry. Mean and S.D. of three independent experiments performed in triplicate are shown; NS, not significant

Article Snippet: 44 Western blot analysis Western blot analysis was performed as described previously 45 using the following antibodies: rabbit anti-RIP3 (Novus Biologicals, Littleton, CO, USA), rabbit anti-MLKL (GeneTex, Irvine, CA, USA, for human or Sigma-Aldrich for mouse), mouse anti-caspase-8 (Enzo Life Sciences), rabbit anti-Prx3 (Abcam, Cambridge, MA, USA), rabbit anti-Bak (BD Biosciences, rabbit anti-Bax (Millipore, Darmstadt, Germany), mouse anti- β -actin (Sigma-Aldrich) or mouse anti-GAPDH (Biotrend, Cologne, Germany) as loading control and secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Flow Cytometry, Staining

Journal: Cell Death and Differentiation

Article Title: A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis

doi: 10.1038/cdd.2016.102

Figure Lengend Snippet: ROS production is required for BV6/Dexa-induced Bak activation, loss of MMP and cell death. (a) Cells were treated for 6 h with BV6 (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) and 200 μM Dexa in the presence or absence of 100 μM MnTBAP or 100 μM α-Tocopherol, Jurkat cells were additionally treated with 20 μM zVAD.fmk. Bak activation was determined by immunoprecipitation using an active conformation-specific antibody. Protein expression of Bak was analyzed by western blotting. β-Actin served as loading control. (b) Tanoue cells were treated for 4 h with 3 μM BV6 and 200 μM Dexa, treatment with 2 μM auranofin for 40 min served as positive control for ROS-mediated oxidative modifications. Oxidative thiol modifications of Bak (oxidized) were analyzed by BIAM switch assay. Oxidized Prx3 served as marker for oxidative stress. Total protein levels of Bak and Prx3 remained unchanged upon treatment. (c) Cells were treated for 24 h with BV6 (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) and/or 200 μM Dexa in the presence or absence of 100 μM MnTBAP or 100 μM α- Tocopherol, Jurkat cells were additionally treated with 20 μM zVAD.fmk. Cell death was determined by FSC/SSC analysis and flow cytometry. Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05. (d) Cells were treated for 8 h with BV6 and 200 μM Dexa (Tanoue: 3 μM BV6; Jurkat: 5 μM BV6) in the presence or absence of 100 μM MnTBAP or 100 μM α-Tocopherol, Jurkat cells were additionally treated with 20 μM zVAD.fmk. Loss of MMP was assessed by TMRM staining and flow cytometry. Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05

Article Snippet: 44 Western blot analysis Western blot analysis was performed as described previously 45 using the following antibodies: rabbit anti-RIP3 (Novus Biologicals, Littleton, CO, USA), rabbit anti-MLKL (GeneTex, Irvine, CA, USA, for human or Sigma-Aldrich for mouse), mouse anti-caspase-8 (Enzo Life Sciences), rabbit anti-Prx3 (Abcam, Cambridge, MA, USA), rabbit anti-Bak (BD Biosciences, rabbit anti-Bax (Millipore, Darmstadt, Germany), mouse anti- β -actin (Sigma-Aldrich) or mouse anti-GAPDH (Biotrend, Cologne, Germany) as loading control and secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Immunoprecipitation, Expressing, Western Blot, Positive Control, Marker, Flow Cytometry, Staining

Journal: Cell Death and Differentiation

Article Title: A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis

doi: 10.1038/cdd.2016.102

Figure Lengend Snippet: RIP3 and MLKL are required for Bak activation and mitochondrial perturbations during BV6/Dexa-induced necroptosis. Tanoue cells were transiently transfected with two distinct siRNAs targeting RIP3, MLKL or control siRNA, and treated for 6 h with 3 μM BV6 and 200 μM Dexa. MEFs were treated for 12 h with 5 μM BV6 and 200 μM Dexa in the presence of 20 μM zVAD.fmk. (a and b) Bak (a) or Bax (b) activation was determined by immunoprecipitation using active conformation-specific antibodies. Protein expression of Bak, Bax, RIP3 and MLKL were analyzed by western blotting. β-Actin served as loading control. (c and d) Loss of MMP was determined by TMRM staining and flow cytometry (c) or ImageXpress Micro XLS system (d). Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05. (e and f) ROS production was determined by CellROX staining and flow cytometry (e) or ImageXpress Micro XLS system (f). Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05. (g and h) Respiration was determined by the Oxygraph system. Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05

Article Snippet: 44 Western blot analysis Western blot analysis was performed as described previously 45 using the following antibodies: rabbit anti-RIP3 (Novus Biologicals, Littleton, CO, USA), rabbit anti-MLKL (GeneTex, Irvine, CA, USA, for human or Sigma-Aldrich for mouse), mouse anti-caspase-8 (Enzo Life Sciences), rabbit anti-Prx3 (Abcam, Cambridge, MA, USA), rabbit anti-Bak (BD Biosciences, rabbit anti-Bax (Millipore, Darmstadt, Germany), mouse anti- β -actin (Sigma-Aldrich) or mouse anti-GAPDH (Biotrend, Cologne, Germany) as loading control and secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Immunoprecipitation, Expressing, Western Blot, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Attenuation of Apoptosis Underlies B Lymphocyte Stimulator Enhancement of Humoral Immune Response

doi:

Figure Lengend Snippet: Regulation of Bcl-2 family proteins by BLyS. (A) Expression of Bak and Bcl-xL in high density (H) and low density (L) splenic B cells isolated from mice administered with BLyS or PBS on day 11 of immunization with NP-CGG. (B) Expression of Bak, Bcl-xL, and Bcl-2 on high density splenic B cells, either freshly isolated (lane 1), after 24 h of in vitro culture with media alone (lanes 2 and 3), or after 48 h of coculture with CD40L cells (lanes 4 and 5), in the presence or absence of BLyS. ns, nonspecific bands used as controls for sample loading.

Article Snippet: Proteins (15–40 μg, in equal amounts) in whole cell extracts were resolved by electrophoresis on a 12% polyacrylamide-SDS gel, transferred to a polyvinylidine difluoride membrane (PVDF; Millipore), and probed with the following antisera: hamster anti–mouse Bcl-2, rabbit anti–human Bcl-xL, and rabbit anti–human Bak (BD PharMingen).

Techniques: Expressing, Isolation, In Vitro

Journal:

Article Title: DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells

doi: 10.1101/gad.1156903

Figure Lengend Snippet: BAK and MCL-1 form a complex that is disrupted during adenovirus infection. Immunoprecipitation (IP) of BAK and MCL-1 from mock, Ad5dl309, Ad5dl337 infected cells was carried out with anti-p19, anti-PCNA, anti-BAK(Ab-1), anti-BAK(TM), anti-MCL-1, anti-MCL-1(Ab-1) antibodies (Abs) from the soluble fraction of cells lysed in CHAPS-containing buffer at 24 h post-infection. Mock-infected HeLa cell lysate (M) was utilized as a marker for MCL-1, BAK, and BAX expression levels. Western blotting was carried out on precipitated material with an anti-MCL-1 antibody, anti-BAK plus anti-BAX antibodies, or an anti-E1B 19K antibody. Samples of lysates collected before IP (Lysates) were analyzed to ascertain total protein levels prior to immunoprecipitation.

Article Snippet: Immunoprecipitations were carried out with the following primary antibodies: anti-p19 rabbit polyclonal and anti-PCNA mouse monoclonal (Santa Cruz Biotechnology); anti-BAK(Ab-1) mouse monoclonal (Oncogene Research); anti-BAK (minus transmembrane domain, or -TM) rabbit polyclonal (PharMingen); anti-BAK-NT rabbit polyclonal (Upstate Biotechnology); anti-MCL-1 rabbit polyclonal (Stressgen); and anti-MCL-1 mouse monoclonal (Oncogene Research); anti-MYC rabbit polyclonal (Santa Cruz Biotechnology); anti-BAX(11-30) rabbit polyclonal (Santa Cruz Biotechnology); anti-BAX (150-165) rabbit polyclonal (Oncogene Research); anti-BAX(43-61) rabbit polyclonal (PharMingen); and anti-BAK G-23 rabbit polyclonal (Santa Cruz Biotechnology).

Techniques: Infection, Immunoprecipitation, Marker, Expressing, Western Blot