Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Schematic representation of the domain structures of mitophagy receptor proteins, highlighting the LIR motifs (pink color) and the C-terminal transmembrane (TM) domain (blue color). (B) Clustal Omega alignment of BCL2L13 orthologs across species showing conservation of reported phosphorylation sites. Note that S261 lies at the start of the second of two repeat elements (purple hue) which precede S275 and the LIR motif (pink hue). Reported phosphorylation sites in mass spectrometry datasets are highlighted in yellow. (C) Immunoblots of WT (NT) or CRISPR/Cas9 RNP-BCL2L13 knockout (KO) MEF cell lines bearing stable lentiviral expression of indicated BCL2L13 cDNAs, treated with 20 µM CCCP for 1 hour. These blots demonstrate that the phospho-specific antibodies detect BCL2L13 phosphorylation specifically at serine 261 (S261) and serine 275 (S275) in MEF cells, with phosphorylation levels increasing following CCCP treatment. Arrows indicate endogenous mouse Bcl2l13 (predicted to be 7 kDA smaller versus human). (D) Phosphorylation of endogenous Bcl2L13 on Ser261 and Ser275 compared to the regulation of other mitophagy receptor protein levels. Immunoblots with indicated antibodies on WT or BCL2L13 KO MEF cells, treated with DMSO vehicle (veh) or 10 µM 991 or 20 µM CCCP or 1 µM rotenone or 1mM DFP. (E) Quantification of BCL2L13 Ser 261 (left) and BCL2L13 Ser 275 (right) from the immunoblot shown in panel D. Densitometric analysis was performed to assess protein expression levels. Phospho-protein levels were quantified and normalized to total protein levels.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Phospho-proteomics, Mass Spectrometry, Western Blot, CRISPR, Knock-Out, Expressing

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Immunoblot of U2OS cells stably expressing flag-tagged BCL2L13, treated with various autophagy and mitophagy-inducing compounds: 20 µM CCCP, 1 mM DFP, 10 µM 991, 100 ng/ml rotenone, and 1 µM INK128 for 1 hour. After treatment, cells were lysed in 2% CHAPS buffer, and the lysates were subjected to flag immunoprecipitation (IP). (B) Protein sequence coverage map of the BCL2L13 samples submitted for mass spectrometry analysis. GluC digestion resulted in 75% coverage of the BCL2L13 protein sequence. (C) The table summarizes the spectral counts for each detected phosphorylation site. Notably, three phosphorylation sites — S275, S259, and S261 — show a significant increase in spec count after CCCP treatment compared to DMSO vehicle. These sites match the consensus kinase motifs for ULK1- or TBK1-like kinases, as highlighted in the red box. (D) Immunoblots of MEF cells from wild-type (WT) or CRISPR-Cas9 RNP-mediated BCL2L13 knockout (KO) lines, treated with 20 µM CCCP at the indicated time-point to validate the phospho-specific antibody developed by CST. These blots demonstrate that the antibody specifically detects BCL2L13 at serine 275 (S275) in MEF cells, with phosphorylation levels increasing following treatment with CCCP.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Sequencing, Mass Spectrometry, Phospho-proteomics, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: ( A ) Diagram of the Mito-QC construct: mCherry-GFP tandem mitophagy assay. ( B) Representative histogram of ratiometric flow cytometry analysis before and after 4 hours of CCCP in WT MEF cells stably expressing the mitophagy reporter Mito-QC (“MEF-QC”). (C) MEF-QC cells were treated with CCCP or DFP at the indicated time points. Following treatment, cells were harvested in flow buffer, and mitophagy was quantified using flow cytometry based on the mCherry/GFP ratio. ( D ) HEK293A-QC cells treated and analyzed as in panel C. (E) Immunoblot of protein levels of the mitophagy receptors in MEF and HEK293A following CCCP or DFP at the indicated timepoints. ( F) Human and mouse BCL2L13 CRISPR-Cas9 deletion was assessed in MEF-QC and HEK293A-QC cells. (G) WT or CRISPR-Cas9 mediated BCL2L13 knockout (KO) HEK293-QC cells were treated with CCCP or DFP at the indicated time points. Following treatment, cells were harvested in flow buffer, and mitophagy was quantified using flow cytometry based on the mCherry/GFP ratio. ( H) WT or BCL2L13 KO MEF-QC cells were treated with CCCP or DFP at the indicated time points and mitophagy was quantified using flow cytometry based on the mCherry/GFP ratio as in panel G. (I) Immunoblot analysis of mitophagy receptors in WT and BCL2L13 KO MEFs reveals differential regulation of ubiquitin-independent mitophagy receptors MEF cells exhibit differential regulation of the ubiquitin-independent mitophagy receptors. (J) Immunoblot analysis of mitophagy receptors in WT and BCL2L13 KO HEK293A cells. Ratiometric flow cytometry data (mCherry/GFP ratio) are presented as the mean ± SEM for biological replicates (N = 3). Statistical significance was assessed using Two-Way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001).

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Construct, Mitophagy Assay, Flow Cytometry, Stable Transfection, Expressing, Western Blot, CRISPR, Knock-Out, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Immunoblots of MEF cells from wild-type (WT) or CRISPR-Cas9 RNP-mediated TBK1 knockout (KO) lines, treated with vehicle or TBK1 activator LPS for 1 hour. (B) Immunoblot analysis of wild-type (WT) MEFs, TBK1 knockout (KO) MEFs, and AMPK knockout (AMPKDKO) MEFs treated with vehicle (Veh), LPS, CCCP, or MK8722. (C) Quantification of BCL2L13 Ser 261 (left) and BCL2L13 Ser 275 (right) from the immunoblot shown in B. Densitometric analysis was performed to assess protein expression levels. Phospho-protein levels were quantified and normalized to total protein levels and loading control, Actin.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Western Blot, CRISPR, Knock-Out, Expressing, Control

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: (A) Immunoblot analysis of CRISPR/Cas9 knockouts of individual mitophagy receptors, and triple and quadruple deletions in MEFs. (B) MitoQC functional mitophagy assay after CCCP in MEFs from panel A, as analyzed by flow cytometry. Quantitative analysis was performed on the ratiometric flow cytometry data (mCherry/GFP). Data are presented as mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA. Asterisks indicate significant differences compared to the control group (* p < 0.05). (C) Representative confocal micrographs of Control (NT), BCL2L13 KO, Triple KO, and Quad KO MEFs treated with 20 µM CCCP for the indicated timepoints and immunostained for HSP60 and Rab7. Zoomed-in regions highlight co-localization of HSP60 and Rab7 (indicated by blue arrows). (D) Mitolysosome formation was quantified as RAB7+ mitochondrial fragments using CellProfiler. Data points represent experimental averages of more than 200 cells per condition (n = 3). Statistical significance was determined using one-way ANOVA. Asterisks indicate significant differences compared to the control group (* p < 0.05). (E) Model for the regulation of BCL2L13. After mitochondrial damage, AMPK activates Parkin and BCL2L13, while simultaneously suppressing NIX and BNIP3 activation by ULK1. In contrast, under hypoxic conditions, ULK1-dependent phosphorylation and activation of BNIP3, NIX, and FUNDC1 are critical events, and neither Parkin nor BCL2L13 appears to be involved. Thus, Bcl2L13 is currently the only ubiquitin-independent mitophagy receptor known to be acutely activated by mitochondrial damage.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Western Blot, CRISPR, Functional Assay, Mitophagy Assay, Flow Cytometry, Control, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation

doi: 10.1101/2025.07.10.663832

Figure Lengend Snippet: Using MitoQC flow cytometry analysis and quantitative co-locatization of HSP60 and RAB7 by high-throughput indirect immunofluorescence imaging, as in . (A) Endogenous mouse BCL2L13 levels (NT cells) alongside stable expression of WT human Flag-tagged BCL2L13 and Serine-to-Alanine mutants in the Quad KO MEFs. Endogenous BCL2L13 protein levels are comparable to the flag-tagged addbacks. Note that human BCL2L13 runs ∼20kD larger than mouse BCL2L13. (B) Mitophagy in Quad KO MEFs stably expressing the mitophagy reporter (mito-QC) with addback of BCL2L13 WT point mutants was analyzed using flow cytometry. Quantitative analysis of ratiometric flow cytometry (mCherry/GFP). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. Asterisks indicate statistically significant differences compared to the control group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001). (C) Control (NT), Triple KO, and Quad KO MEF stably expressing with indicated BCL2L13 cDNAs were treated with 20 µM CCCP for 1H or 4H timepoints and immunostained for HSP60 and Rab7, as detailed in . Mitolysosome formation was quantified as RAB7+ mitochondrial fragments using CellProfiler. Bar graphs represent the mean and standard error of >200 cells/condition.

Article Snippet: The cDNA encoding human BCL2L13 was obtained from OriGene (RC200042).

Techniques: Flow Cytometry, High Throughput Screening Assay, Immunofluorescence, Imaging, Expressing, Stable Transfection, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: (A) QRT-PCR receptor levels of Alk1 in the cell lines indicated (left panel). Human microvascular endothelial cells served as the positive control for Alk1 expression . Side-by-side comparison of Alk1, 2, 3, and 6 in PA1 and P76 (right panel). (B-C) Western blotting of either normal breast cell line MCF10A and FTEC (P211) or (C) transformed breast and ovarian cancer cells in the presence of increasing doses of GDF2 for 30 minutes as indicated. Total SMAD1/5 served as loading control. (D) Western blotting for pSMAD2/3 in P76 cells treated with GDF2 (10 ng/ml) for the indicated times. Lysates for TGF-β–treated cells were used as a positive control. (E) GDF2 specifically induces BRE-luciferase promoter activity. Luciferase activity normalized to untreated HEK293 cells transfected with either BRE-Luc, PE2.1 Luc, or P3tP Luc reporter plasmids treated with BMP2 (10 nM), GDF2 (10 ng/ml), or TGFβ (100 pM) for 24 hours is presented.

Article Snippet: Total SMAD1/5 (#sc-6201), ID1 (#365654), and GDF2 (#130703) were from Santa Cruz Biotech.

Techniques: Quantitative RT-PCR, Positive Control, Expressing, Western Blot, Transformation Assay, Luciferase, Activity Assay, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: GDF2 activates SMAD1/5 in breast and ovarian epithelial cells. (A) Western blotting of lysates from untransformed breast (MCF10A) and FTEC line FTEC 211 treated with GDF2 (10 ng/ml) for the indicated times and immunoblotted for pSMAD1/5 and SMAD1/5. (B) Western blotting of lysates from transformed breast cancer lines 4T1 and 67NR including the transformed oncogenic FTEC P76 and ovarian cancer line PA1 treated as in (A) and immunoblotted for pSMAD1/5 and total SMAD1/5. (C) SMAD1/5 translocates to the nucleus. Immunofluorescence images of 4T1 cells treated with GDF2 for 30 minutes followed by immunostaining for SMAD1/5. Overlay images are shown with the nuclear stain 4′6-diamidino-2-phenylindole. (D) GDF2 specifically induces BRE-luciferase promoter activity. Luciferase activity (Methods) of either BMP4 (10 nM)– or GDF2 (10 ng/ml)–treated MCF10A, p76, and PA1 cells as indicated normalized to untreated cells is presented. (E) ID1 target protein expression. Western blotting for ID1 in 4T1 and 67NR cells treated with GDF2 at indicated time points. Loading control was actin. (F) pSMAD1/5 phosphorylation in response to BMP10 is less robust compared with GDF2. Western blotting of MCF10A and PA1 lysates treated with 10 ng/ml of GDF2 or BMP10 for the indicated times.

Article Snippet: Total SMAD1/5 (#sc-6201), ID1 (#365654), and GDF2 (#130703) were from Santa Cruz Biotech.

Techniques: Western Blot, Transformation Assay, Immunofluorescence, Immunostaining, Staining, Luciferase, Activity Assay, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: (A) QRT-PCR analyses of receptor levels in breast (MCF10A and 4T1) and ovarian (PA1 and P76) cell lines. (B) Western blotting in MCF10A cells, as indicated in the presence and absence of SB431542 3 μM (+) or 5 μM (++) for 30 minutes −/+ TGFβ. (C) Quantification of pSMAD1/5 levels in Figure 2 B represented as fold change in intensities normalized to total SMAD1/5 levels.

Article Snippet: Total SMAD1/5 (#sc-6201), ID1 (#365654), and GDF2 (#130703) were from Santa Cruz Biotech.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: ALK3 and ALK6 are required for GDF2-induced SMAD1/5 phosphorylation. (A-B) Western blotting for pSMAD1/5 activation in PA1 and MCF10A cells in the presence and absence of dorsomorphin 1 μM (+) or 3 μM (++), SB431542 3 μM (+) or 5 μM (++), or ML347 500 nM (+) or 1 mM (++) as indicated with and without GDF2 (10 ng/ml) as indicated (quantification of pSMAD1/5 levels presented in Supplementary Figure S2 C ). (C) Immunoblotting of pSMAD1/5 in PA1 cells in the presence of shRNAs to ALK2, ALK3, ALK6, and BMPRII without and with GDF2 treatment (10 ng/ml) for 30 minutes. (D) QRT-PCR analyses of (C) to confirm reduced expression of ALK2, ALK3, ALK6, and BMPRII expression as indicated. (E) Kinase inactive ALK3 and ALK6 inhibit SMAD1/5 phosphorylation. Western blotting as indicated in MCF10A and PA1 cells in the presence of either mock transfected or HA-tagged kinase inactive ALK3 (ALK3 K-R) or ALK6 (ALK6 K-R) and treated with GDF2 for the time points indicated. Actin was the loading control. (F) Dorsomorphin inhibits SMAD1/5 transcriptional activation. BRE-luciferase reporter activity in indicated cells in the absence (GDF2 alone) or presence of 1 μM dorsomorphin (GDF2+DM). Fold induction of luciferase activity compared with DMSO-treated control cells is presented.

Article Snippet: Total SMAD1/5 (#sc-6201), ID1 (#365654), and GDF2 (#130703) were from Santa Cruz Biotech.

Techniques: Western Blot, Activation Assay, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: GDF2 stimulates anoikis. (A) Indicated cell lines were plated for anoikis as described in Methods on poly-HEMA–coated plates and treated with either 10 ng/ml (+) or 20 ng/ml (++) of GDF2. Western blotting for CC3 and actin is presented. Normalized densitometry analysis of CC3 intensities is presented below. (B) Absorbance values using the MTT assay (Methods) after anoikis for 24 hours with different concentrations of GDF2 are presented. Data are representative of two independent experiments (* P < .05 and **** P < .0001 in p76 and 4T1 cells, respectively). Error bars indicate SEM. (C) Acini size of MCF10A cells grown on growth factor reduced Matrigel with or without 20 ng/ml of GDF2 in the presence or absence of pan-caspase inhibitor Z-VAD-FMK for 10 days is presented. Mean acini width is quantified (*** P < .01). N = 30; error bars indicate SEM. (D) Western blotting of same cell lysates prepared as described for (A) was probed for pSMAD1/5 and total SMAD1/5. Actin was the loading control.

Article Snippet: Total SMAD1/5 (#sc-6201), ID1 (#365654), and GDF2 (#130703) were from Santa Cruz Biotech.

Techniques: Western Blot, MTT Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia 1

doi: 10.1016/j.neo.2015.11.003

Figure Lengend Snippet: Anoikis susceptibility is mediated via ALK3/ALK6. (A) Western blotting of 4T1 cells grown under anoikis conditions either with or without 1 μM dorsomorphin and GDF2 as indicated. Lysates were immunoblotted against CC3. Actin was the loading control (quantification of CC3 levels presented in Supplementary Figure S3 E ). (B) Kinase mutants ALK3 K-R and ALK6 K-R reduce anoikis. Western blotting of PA1 cells expressing control, ALK3 K-R, or ALK6 K-R was plated for anoikis (Methods) in the absence or presence of GDF2 for indicated times (quantification of CC3 levels presented in Supplementary Figure S3 F ). (C) MTT absorbance values of PA1 cells expressing shScr, shALK3 or shALK6 after 24 hours of anoikis in the absence and presence of 10 ng/ml of GDF2 are presented. Error bars indicate SEM. (D) Western blotting of same cells as in (C) harvested after 24 hours of anoikis and immunoblotted for CC3. Actin was the loading control. (E) Western blotting as indicated of PA1 cells expressing shSMAD1 or shScr at the indicated times under anoikis conditions (quantification of CC3 presented in Supplementary Figure S3 G ). SMAD1 transcript levels upon shRNA to SMAD1 relative to controls are presented in the adjacent graph. Actin was the loading control.

Article Snippet: Total SMAD1/5 (#sc-6201), ID1 (#365654), and GDF2 (#130703) were from Santa Cruz Biotech.

Techniques: Western Blot, Expressing, shRNA

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A) Structures of human Bcl-rambo and its mutants. (B) S2 cells were transfected with pMT-V5-His A, pMT-V5-His A/ Bcl-rambo , or pMT-V5-His A/ Bcl-rambo (ΔTM) and then incubated in the presence of CuSO 4 (500 μM) and Z-VAD-fmk (20 μM) for 24 h. S2 cells were stained for Bcl-rambo (green) and with DAPI (blue) and MitoTracker ® Red (red). The stained cells in at least five different fields were observed by confocal laser scanning microscopy. Optical sections containing single transfected cells are shown. Data were representative of two independent experiments. Scale bars indicate 10 μm. (C) S2 cells were transfected with pMT-V5-His A, pMT-V5-His A/ Bcl-rambo , or pMT-V5-His A/ Bcl-rambo (ΔTM) and incubated in the presence of CuSO 4 (500 μM) for 24 h. S2 cells were homogenized and separated into PNL, P10, P100, and S100 fractions. The fractions were analyzed by Western blotting using anti-Bcl-rambo, anti-HSP60, and anti-α-tubulin antibodies. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Transfection, Incubation, Staining, Confocal Laser Scanning Microscopy, Western Blot

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A) S2 cells were transfected with pAct5C- GAL4 together with pUAST, pUAST- Bcl-rambo , pUAST- Bcl-rambo (ΔTM) , pUAST- Bcl-rambo (TM) , or pUAST- DsRed-monomer for 24 h. Cells were stained with Hoechst 33342. Nuclear morphology was observed by fluorescent microscopy. Apoptotic cells (%) are shown as the mean ± S.E. of three independent experiments. ** P <0.01, significantly different from the control. n.s., not significant. Transfection efficiency was measured by counting DsRed-monomer-expressing cells, and calculated to be 17.0 ± 0.7% (the mean ± S.E of three independent experiments). (B) S2 cells were transfected with (+) or without (–) pMT-V5-His A/ Bcl-rambo (no tag), or pMT-V5-His A/ Drob-1 (FLAG tag) for 20 h and then incubated in the presence of CuSO 4 (500 μM) for the indicated times. The expression of Bcl-rambo and Drob-1 was analyzed by Western blotting using anti-Bcl-rambo and anti-FLAG antibodies, respectively. Data were representative of three independent experiments. (C) S2 cells were transfected with pMT-V5-His A (open circles), pMT-V5-His A/ Bcl-rambo (filled circles), or pMT-V5-His A/ Drob-1 (filled squares) for 20 h and then incubated in the presence of CuSO 4 (500 μM) for the indicated times. Cells were stained with Hoechst 33342. Apoptotic cells (%) are shown as the mean ± S.E. of three independent experiments. ** P <0.01, significantly different from the control. (D) S2 cells were transfected with (+) or without (–) pMT-V5-His A/ Bcl-rambo or pMT-V5-His A/ Drob-1 for 20 h and then incubated in the presence of CuSO 4 (500 μM) for 8 h. The cytosolic fraction and organelle fraction containing mitochondria were both analyzed by Western blotting using anti-cytochrome c (Cyt. c ), α-tubulin, and anti-HSP60 antibodies. Data are representative of two independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Transfection, Staining, Microscopy, Control, Expressing, FLAG-tag, Incubation, Western Blot

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using dpp - GAL4 driver fly lines. (a) w ; +; dpp-GAL4 /+, (b) w ; UAS-Bcl-rambo /+; dpp-GAL4 /+, (c) w ; UAS-Bcl-rambo(ΔTM)/+ ; dpp-GAL4 /+. The morphology of the wings was observed under light microscopy. The white arrow indicates morphological aberrations in the wing vein. The L3-L4 area was measured by Image J software (B). Data are shown as the mean ± S.E. of three independent experiments (n = 4–5 for each experiment). ** P <0.05, significantly different from control. n.s., not significant. (C) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using pnr - GAL4 driver lines. (a) w ; +; pnr-GAL4 /+, (b) w ; UAS-Bcl-rambo /+; pnr-GAL4 /+, (c) w ; UAS-Bcl-rambo (ΔTM)/+ ; pnr-GAL4/+ . The morphology of adult thoraxes was observed by SEM. Scale bars indicate 100 μm. Data were representative of three independent experiments. (D and E) Cell lysates of third instar larval salivary glands were prepared from sg-GAL4/w ; +; +, sg-GAL4/w ; UAS-Bcl-rambo/+ ; +, and sg-GAL4/w ; UAS-Bcl-rambo (ΔTM)/ +; + flies, and analyzed by Western blotting. The amount of Bcl-rambo and Bcl-rambo (ΔTM) was normalized to that of α-tubulin. The protein expression of Bcl-rambo (%) is shown as the mean ± S.E. of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy, Software, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR - GAL4 driver fly lines. (a, e, i) GMR-GAL4 / w ; +/ CyO ; +, (b, f, j) GMR-GAL4 / w ; UAS-GFP /+; +, (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO ; +, (d, h, l) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) / CyO ; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Light microscopy and SEM photographs were taken from different individuals. Data were representative of three independent experiments. (B) Eye imaginal discs were stained for Bcl-rambo (green) and with Hoechst 33342 (blue). (a and d) GMR-GAL4 / w ; +/ CyO or Sp ; +, (b and e) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO or Sp ; +, (c and f) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM) / CyO or Sp ; +. White arrows show the morphogenetic furrow. Scale bars indicate 50 μm. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy, Staining

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) (a, d) GMR-GAL4 / w ; +/ CyO or Sp ; +, (b, e) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO or Sp ; +, (c, f) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) / CyO or Sp ; +. The eye imaginal discs were labeled for activated caspase-3/7 (green) and with Hoechst 33342 (blue). Scale bars indicate 50 μm. White arrows indicate the morphogenetic furrow. The number of fluorescent cells harboring activated caspases from the morphogenetic furrow to the posterior region of imaginal eye discs was measured (B). Data are shown as the mean ± S.E. of three independent experiments (n = 6 for each experiment). ** P <0.01, significantly different from control. n.s., not significant.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Labeling, Control

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR - GAL4 driver fly lines. (A) (a, e, i) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, f, j) GMR-GAL4 / w ; UAS-Bcl-rambo /+; UAS-p35 /+, (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo /+; UAS-Diap1 /+, (d, h, l) GMR-GAL4/w ; UAS-Bcl-rambo/+ ; GMR-Diap2/+ . (B) (a, e, i) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; +, (b, f, j) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; UAS-p35/+ , (c, g, k) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; UAS-Diap1/+ , (d, h, l) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; GMR-Diap2/+ . The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Light microscopy and SEM photographs were taken from different individuals. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Pupal retinae 42 h after pupal formation were stained with an anti-discs large antibody (green). (a) w/+ ; +; +, (b) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (c) GMR-GAL4 / w ; UAS-Bcl-rambo /+; UAS-p35 /+, (d) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (e) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; UAS-p35 /+. Scale bars indicate 10 μm. The numbers of normal and aberrant ommatidia were counted. Aberrant morphology (%) is shown as the mean ± S.E. of three independent experiments (n = 5 for each experiment). ** P <0.01. n.s., not significant.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Eye imaginal discs were stained for DAPI (blue) and Elav (green), together with Prospero (red) (A) or Cut (red) (B). (a–d) GMR-GAL4 / w ; +/ CyO or Sp ; +, (e–h) GMR-GAL4 / w ; UAS-Bcl-rambo / CyO or Sp ; +, (i–l) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM) / CyO or Sp ; +. Scale bars indicate 10 μm. Data were representative of at least two independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A and B) Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR - GAL4 driver fly lines. (A) (a, f, k) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, g, l) GMR-GAL4 / w ; UAS-Bcl-rambo / Drob-1 E26 ; +, (c, h, m) GMR-GAL4 / w ; UAS-Bcl-rambo / Drob-1 W105 ; +, (d, i, n) GMR-GAL4/w ; UAS-Bcl-rambo/Drob-1 E26 Buffy H37 ; +, (e, j, o) GMR-GAL4/w ; UAS-Bcl-rambo/Drob-1 W105 Buffy H37 ; +. (B) (a, f, k) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/+ ; +, (b, g, l) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 E26 ; +, (c, h, m) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 W105 ; +, (d, i, n) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 E26 Buffy H37 ; +, (e, j, o) GMR-GAL4/w ; UAS-Bcl-rambo (ΔTM)/Drob-1 W105 Buffy H37 ; +. The morphology of the adult eyes was observed by light microscopy (a–e) and SEM (f–o). Scale bars in f–j and k–o indicate 50 μm and 14.2 μm, respectively. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: HEK293T cells were transfected with (+) or without (–) expression vectors for FLAG-tagged Bcl-rambo or FLAG-tagged Buffy together with VSV-tagged Buffy or VSV-tagged Drob-1/Debcl for in the presence of zVAD-fmk (20 μM) for 16 h. Cell lysates were immunoprecipitated with anti-FLAG antibody-conjugated beads. Immunoprecipitates (IP) were analyzed by Western blotting using an anti-VSV antibody. Cell lysates were analyzed by Western blotting using anti-FLAG, anti-VSV, and β-actin antibodies. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

doi: 10.1371/journal.pone.0157823

Figure Lengend Snippet: (A to C) (A) (a, d, g) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, e, h) sesB org / GMR-GAL4 ; UAS-Bcl-rambo /+; +, (c, f, i) w 67c23 P{lacW}Ant2 G0247 sesB G0247 / GMR-GAL4 ; UAS-Bcl-rambo /+; +. (B) (a, d, g) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (b, e, h) sesB org / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +, (c, f, i) w 67c23 P{lacW}Ant2 G0247 sesB G0247 / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +. The morphology of adult eyes was observed by light microscopy (a–c) and SEM (d–i). (C) (a, e, i) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, f, j) w 67c23 P{lacW}schlank G0061 schlank G0061 / GMR-GAL4 ; UAS-Bcl-rambo /+; + (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (d, h, l) w 67c23 P{lacW}schlank G0061 schlank G0061 / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Data were representative of two independent experiments. (D) (a, e, i) GMR-GAL4 / w ; UAS-Bcl-rambo /+; +, (b, f, j) w 1118 P{EP}Atg8a EP362 / GMR-GAL4 ; UAS-Bcl-rambo /+; + (c, g, k) GMR-GAL4 / w ; UAS-Bcl-rambo (ΔTM) /+; +, (d, h, l) w 1118 P{EP}Atg8a EP362 / GMR-GAL4 ; UAS-Bcl-rambo (ΔTM) /+; +. The morphology of adult eyes was observed by light microscopy (a–d) and SEM (e–l). Scale bars in e–h and i–l indicate 50 μm and 14.2 μm, respectively. Data were representative of three independent experiments.

Article Snippet: Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained.

Techniques: Light Microscopy