EMBO/soda-nel huggingface dataset:
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf7

CF human nasal mucosae cultured with cystamine or NAC (patients 4–10 in Supplementary Information, Table S1) (a–c) in the presence or absence of 3-MA (patients 6–10 in Supplementary Information, Table S1) (b, c). (a) Immunoblot of beclin 1. (b) Fluorescence microscopy quantification of LC3-positive dots. Results represent means ± s.d. Asterisk, P 0.05 between groups of treatment; ANOVA. (c) Confocal microscopy micrographs of p62. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative of five patients per group. (d–i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e–i) (n = 7 per group). (d) Immunoblot of beclin 1. (e) Quantification of LC3 dots (per mm2 of lung tissue). Means ± s.d. Asterisk, P 0.01; ANOVA. (f) Immunoblot of TG2. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (g) Confocal microscopy of SUMO-1 in lung tissues. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. (h) Number of CD68+ macrophages counted in 15–20 different randomly taken sections. Means ± s.d. per mm2 of lung tissue from seven mice in each group. Asterisk, P 0.01; ANOVA. (i) MPO activity in lung homogenates (expressed as 10−3 × optical density (OD) s−1). Means ± s.d. for lung tissues from seven mice in each group. Asterisk, P 0.05; ANOVA. (j–m) CftrF508del mice (n = 3 per group) administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (j) TG2 Immunoblot in lung tissues. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (k) Confocal microscopy of TG2 activity. Representative stainings from at least five airway epithelial areas per mouse from three mice per group. L, lumen. Scale bar, 10 μm. (l) Number of CD68+ macrophages counted in 15–20 different randomly taken sections. Means ± s.d. per mm2 of lung tissue. Asterisk, P 0.01; ANOVA. (m) MPO activity in lung homogenates (expressed as 10−3 × OD s−1). Means ± s.d. for lung tissues. Asterisk, P 0.05; ANOVA. Anti-β-Tubulin was used as loading control for immunoblot analysis. Densitometric analysis of blots is shown in Supplementary Information, Fig. S9.
Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition (2010)
figf8

CftrF508del mice (a–d) and Scnn1b-Tg mice (e–k) treated with NAC or PBS (n = 10 per group). (a) Confocal microscopy of LC3 (top) and p62 (bottom) in lung tissues from CftrF508del mice. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative images of ten PBS-treated and ten NAC-treated mice. (b) Quantification of LC3 dots (per mm2 of tissue) in lung tissues from CftrF508del mice. Means ± s.d. Asterisk, P 0.001 versus PBS-treated mice; ANOVA. (c) Number of macrophages per mm2 of lung tissue from CftrF508del mice (CD68-positive cells in 15–20 different randomly taken sections for each mouse lung for each condition); means ± s.d. for three separate experiments. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. (d) MPO activity in lung homogenates from CftrF508del mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. (e) Confocal microscopy of LC3-positive dots in lung tissues from PBS-treated or NAC-treated Scnn1b-Tg mice. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. (f) Number of LC3-positive dots in lung tissues from Scnn1b-Tg mice. Means ± s.d. Asterisk, P 0.01 versus PBS-treated mice; ANOVA. (g) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice. (h) Immunoblot of TG2 and phospho-p42/44 in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice. αβ-Tubulin was used as loading control. (i) Lung histology of PBS-treated and NAC-treated Scnn1b-Tg mice. Haematoxylin staining. Scale bar, 150 μm. Black arrows indicate decrease in lung infiltration in NAC-treated in comparison with PBS-treated mice (arrowheads). (j) Left: confocal microscopy of COX-2 staining (left) in PBS-treated and NAC-treated Scnn1b-Tg mice. Nuclei counterstained with DAPI (blue). Scale bar, 150 μm. Right; number of COX-2-positive cells per mm2 of lung tissue in PBS-treated and NAC-treated Scnn1b-Tg mice. Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. (k) MPO activity in lung homogenates from PBS-treated and NAC-treated Scnn1b-Tg mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. Densitometric analysis of the blot is shown in Supplementary Information, Fig. S9.
Mutating chikungunya virus non-structural protein produces potent live-attenuated vaccine candidate (2019)
Figure 1

Figure 1. R532H and E515V mutations in the nsP region limit CHIKV pathogenicity and alter type-I interferon responses in WT C57BL/6 mice.

(A) Schematic of the CHIKV mutation sites, including CHIKV with R to H (RH), E to V (EV) or both (RHEV) amino acid (a.a) substitutions.

(B) Percentage infectivity of mouse tail fibroblasts (MTFs) infected with WT, RH, RHEV and EV CHIKV (MOI 10, n=3 per group) harvested at 12 hpi. Data are presented as the means ± SEM. Statistical analyses were performed using two-tailed paired T-test (*P =0.019 RH-CHIKV, *P =0.019 RHEV-CHIKV, *P =0.020 EV).

(C, D) (C) Viremia progression and (D) joint inflammation in virus-infected mice was monitored over 2 weeks (n=11 per group). Viremia is detected with CHIKV nsP1 probe via qRT-PCR. Statistical analyses were performed using two-tailed Mann Whitney U test; Viremia (***P =0.000003 RH-CHIKV 1-5 dpi, ***P =0.000065 RH-CHIKV 6 dpi, ***P =0.000281 RH-CHIKV 7 dpi, ***P =0.000079 RH-CHIKV 8 dpi, ***P =0.000275 RHEV-CHIKV 1 dpi, ***P =0.000281 RHEV-CHIKV 2-4 dpi, ***P =0.000054 RHEV-CHIKV 5 dpi, ***P =0.000763 RHEV-CHIKV 6 dpi, ***P =0.000077 RHEV-CHIKV 7 dpi, ***P =0.000595 RHEV-CHIKV 8 dpi, **P =0.007 EV-CHIKV 2 dpi, ***P =0.000011 EV-CHIKV 3 dpi, ***P =0.000034 EV-CHIKV 4 dpi, ***P =0.000394 EV-CHIKV 5 dpi, ***P =0.000128 EV-CHIKV 6 dpi, ***P =0.000006 EV-CHIKV 7 dpi, ***P =0.000054 EV-CHIKV 8 dpi, ***P =0.00017 EV-CHIKV 10 dpi, *P =0.041 EV-CHIKV 12 dpi, *P =0.012 EV-CHIKV 14 dpi), Joint inflammation (***P =0.000003 RH-CHIKV 2-4 dpi, ***P =0.000006 RH-CHIKV 5 dpi, ***P =0.000003 RH-CHIKV 6-8 dpi, ***P =0.000085 RH-CHIKV 9 dpi, ***P =0.000045 RH-CHIKV 10 dpi, ***P =0.000011 RH-CHIKV 11 dpi, **P =0.002 RH-CHIKV 12 dpi, **P =0.004 RH-CHIKV 13 dpi, *P =0.012 RH-CHIKV 14 dpi, ***P =0.000003 RHEV-CHIKV 2-3 dpi, ***P =0.000411 RHEV-CHIKV 4 dpi, **P =0.008 RHEV-CHIKV 5 dpi, ***P =0.000017 RHEV-CHIKV 6 dpi, ***P =0.000094 RHEV-CHIKV 7 dpi, ***P =0.000964 RHEV-CHIKV 8 dpi, **P =0.002 RHEV-CHIKV 11 dpi, ***P =0.029 EV-CHIKV 10 dpi).

(E) Compiled percentages of CHIKV-infected (ZsGreen positive) leukocytes at 2 days post infection (dpi) for the respective groups (n=5 per group). Background ZsGreen signals are indicated by the dotted line. Statistical analyses were performed using two-tailed Mann Whitney U test (*P =0.08 RH-CHIKV, *P =0.008 RHEV-CHIKV, *P =0.008 EV).

(F) Dot-plots of the localized type-I interferon (IFN-α and IFN-β) concentrations at 15 hpi. Data are expressed as the means ± SD. Data were analyzed by one-way ANOVA with post-hoc Tukey test; IFN-α (***P =0.000418 WT-CHIKV versus RH-CHIKV, ***P =0.000688 WT-CHIKV versus RHEV-CHIKV, ***P =0.000407 RH-CHIKV versus EV-CHIKV, **P =0.00122 RH-CHIKV versus PBS, ***P =0.00067 RHEV-CHIKV versus EV-CHIKV, **P =0.002014 RHEV-CHIKV versus PBS), IFN-β (***P <0.000001 WT-CHIKV versus RH-CHIKV, ***P <0.000001 WT-CHIKV versus RHEV-CHIKV, ***P <0.000001 RH-CHIKV versus EV-CHIKV, ***P <0.000001 RH-CHIKV versus PBS, ***P =0.000001 RHEV-CHIKV versus EV-CHIKV, ***P <0.000001 RHEV-CHIKV versus PBS). See also Fig EV1.
Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth (2021)
Figure 1

Figure 1 - Direct molecular interaction between the P5 and AdipoR1.

A Domain architectures of APN and the GST-fused 5-mer peptides (GLYYF; GST-P5 or NIPGL; GST-P10).

B Domain architectures of AdipoR1 and Flag fused full-length AdipoR1 (residue 1-375) or N-terminal-truncated AdipoR1 (residues 89-375). Each construct in the schematic diagram was used for the pulldown experiment.

C GST pulldown of transiently expressed AdipoR1 (residues 1-375) with immobilized GST-P5 or GST-P10 fusion proteins. Pulldown fractions were subjected to western blotting using Flag and GST antibodies.

D GST pulldown of AdipoR1 (residues 1-375) or AdipoR1 (residues 89-375) with GST-P5. The red or blue arrow indicates correct sized bands of AdipoR1 (residues 1-375) or AdipoR1 (residues 89-375), respectively.

E The position of P5 in APN. A stereo ribbon diagram of APN (PDB ID: 4DOU) is drawn, and P5 is shown in magenta sticks.
Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth (2021)
Figure 2

Figure 2 - Both APN and P5 activate AMPK signaling pathway through AdipoR1 in vitro, and P5 induces hair growth factors in DP cells.

A APN-treated ORS or DP cell lysates were analyzed for p-AMPK and p-ERK1/2.

B P5-treated ORS or DP cells lysates were analyzed for p-AMPK and t-AMPK. Densitometric analysis for the ratio of p-AMPK protein to t-AMPK protein (n = 5 (ORS) and 4 (DP) biological replicates / group)

C AdipoR1 siRNA-transfected ORS or DP cells were treated with APN or P5. The cell lysates were analyzed for p-AMPK.

D-H The relative gene expression levels of growth factors in P5-treated DP cells (n = 4 (VEGF, PDGFA), 5 (IGF1), 6 (HGF, FGF7) biological replicates / group)

I-M The relative protein levels of growth factors in P5-treated DP cells (n = 3 biological replicates / group)

Data information: In (B and D-M), One-way ANOVA with Tukey's test compared to each control group. data are presented as the mean ± SEM.
Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth (2021)
Figure 3

Figure 3 - P5 promotes hair growth ex vivo.

A Ex vivo cultured human HF treated with vehicle, P5, or APN for 6 days; scale bar: 1 mm; dotted gray bar: the length of pigmented hair shaft.

B The net length of elongated hair shaft is measured and compared to the control group (n = 38 HFs from 3 independent donors / group); Two-way ANOVA with Dunnett's test compared to the control group.

C IF staining (p-AMPK) of the HF bulb area. Yellow arrow indicates p-AMPK signal; scale bar: 100 µm.

D IF staining (Ki-67) of the HF bulb area; scale bar: 100 µm.

E The numbers of Ki-67-positive cells normalized to DAPI-stained cells (n = 8 HFs / group); One-way ANOVA with Dunnett's test compared to the control group.

Data information: In (B and E), data are presented as the mean ± SEM.
Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth (2021)
Figure 4

Figure 4 - Topical P5 treatment induced the anagen hair cycle through AdipoR1 in vivo.

A Gross morphology of vehicle-, P5-, or minoxidil- treated WT mice.

B Hair cycle scores of vehicle-, P5-, or minoxidil- treated WT mice (n = 16 mice / group); Two-way ANOVA with Dunnett's test compared to the control group.

C H&E and IF for versican of skin tissue; scale bars: 100 µm.

D Anagen induction scores of vehicle-, P5-, or minoxidil- treated WT mice (n = 4 tissues / mice, 4 mice / group); One-way ANOVA with Tukey's test compared to the control group

E Gross morphology of vehicle-, or P5- treated Adipoq-/- mice.

F Hair cycle scores of vehicle-, or P5- treated Adipoq-/- mice (n = 4 mice / group); Two-way ANOVA.

G H&E and IF for versican of skin tissue; scale bars: 100 µm.

H Anagen induction scores of vehicle-, or P5- treated Adipoq-/- mice (n = 6 tissues / mice, 4 mice / group); Unpaired t test

I Gross morphology of vehicle-, or P5- treated Adipor1-/- mice.

J Hair cycle scores of vehicle-, or P5- treated Adipor1-/- mice (n = 5 mice / group); Two-way ANOVA.

K. H&E and IF for versican of skin tissue; scale bars: 100 µm.

L Anagen induction scores of vehicle-, or P5- treated Adipor1-/-mice (n = 6 tissues / mice, 5 mice / group); Unpaired t test

Data information: All values are presented as the mean ± SEM.
Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth (2021)
Figure 5

Figure 5 - Molecular docking simulation followed by mutational studies.

A The most optimal docking model for the AdipoR1-P5 complex. P5 is shown in magenta sticks. Each amino acid of AdipoR1 in yellow was mutated in the following experiments.

B The magnified view for the detailed AdipoR1-P5 complex interface (magenta, P5; yellow, amino acids of AdipoR1); ECL, extracellular loop of AdipoR1.

C-E Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).

F Pulldown of AdipoR1 (residues 89-375) with GST-P5 or GST fused to two mutant peptides (GLYDF and GLYAF). The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).

Data information: In (C-F), data are presented as the mean ± SEM. (One-way ANOVA with Tukey's test compared to the WT or P5).

Expanded View Figure legends
TDP2 negatively regulates axon regeneration by inducing SUMOylation of 
an Ets transcription factor (2019)
Figure 1

Figure 1. Identification of MXL-1 and its involvement in axon regeneration.

A Regulation of svh-2 expression in response to axon injury. Axon injury initiates cAMP signaling and the Ca2+-p38 MAPK pathway, which together function to induce the formation of an ETS-4-CEBP-1 transcription factor complex. This complex binds to the svh-2 promoter to induce svh-2 expression. MXL-1 forms a complex with MDL-1.

B Structure of MXL-1. Schematic diagrams of MXL-1 and its mammalian counterpart Max are shown. The domains (B-HLH-LZ) shown are the basic region (B; blue), helix-loop-helix (HLH; yellow) and leucine zipper (LZ; red). The bold line underneath indicates the extent of the deleted region in the tm1530 deletion mutant.

C Representative D-type motor neurons in wild-type and mxl-1 mutant animals 24 h after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In mutants, proximal ends of axons failed to regenerate (arrowhead). Scale bar = 10 μm.

D Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% confidence intervals (CI).

Data information: In (D), statistical significance was determined by Fisher's exact test; *P < 0.05; ***P <0.001; NS; not significant.
TDP2 negatively regulates axon regeneration by inducing SUMOylation of 
an Ets transcription factor (2019)
Figure 2

Figure 2. MXL-1 is required for the transcriptional induction of the svh-2 gene in response to axon injury.

A Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI.

B Induction of Psvh-2::nls::venus expression in D-type motor neurons by laser surgery. Expression of fluorescent proteins in D-type motor neurons of wild-type, mxl-1, tdpt-1 and mxl-1; tdpt-1 animals 3 h after laser surgery are shown. Yellow arrowheads and white arrows indicate axon and cell bodies, respectively, of D-type neurons. White arrows indicate the sites of laser surgery. D neurons are visualized by CFP under control of the unc-25 promoter. Cell bodies of D-type neurons are magnified and shown within the red boxes. Most of the intestinal fluorescence in these photos is from endogenous and variable background autofluorescence. A schematic representation of D-type motor neurons is shown on the bottom. Scale bars = 10 μm.

Data information: In (A), statistical significance was determined by Fisher's exact test; ***P <0.001; NS; not significant.
TDP2 negatively regulates axon regeneration by inducing SUMOylation of 
an Ets transcription factor (2019)
Figure 3

Figure 3. TDPT-1 negatively regulates axon regeneration.

A, D Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI.

B Interaction of MXL-1 with TDPT-1 by yeast two-hybrid assays. The reporter yeast strain L40u was co-transformed with expression vectors encoding LexA DBD-MXL-1 and GAL4 AD-TDPT-1 as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole for 4 days.

C Structure of TDPT-1. Schematic diagrams of TDPT-1 and human TDP2 are shown. The domains shown are the UBA (red) and Tyrosyl-DNA Phosphodiesterase (TDP; green) domains. The bold line underneath indicates the extent of the deleted region (8 bp) in the km68 mutation. An asterisk indicates a premature stop codon in tdpt-1 caused by the km68 deletion.

Data information: In (A and D), statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.
TDP2 negatively regulates axon regeneration by inducing SUMOylation of 
an Ets transcription factor (2019)
Figure 4

Figure 4. TDPT-1 induces SUMOylation of ETS-4.

A Relationship among MXL-1, TDPT-1, ETS-4, CEBP-1 and PKA in the regulation of axon injury-induced svh-2 expression.

B Interaction between ETS-4 and TDPT-1. COS-7 cells were transfected with plasmids encoding T7-TDPT-1, HA-ETS-4 (WT), HA-ETS-4(S73E) (SE) and HA-ETS-4(S73A) (SA), as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-T7 antibody (IP: T7) were analyzed by immunoblotting (IB).

C In vitro SUMOylation of ETS-4. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands.

D Schematic representation of the two putative consensus SUMOylation sites and domain structure in ETS-4. Domains are shown as follows: a pointed domain (PNT; yellow) and an Ets DNA-binding domain (ETS; blue).

E SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), T7-TDPT-1, His-SUMO-1, His-SUMO-2 and Myc-MXL-1, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).
TDP2 negatively regulates axon regeneration by inducing SUMOylation of 
an Ets transcription factor (2019)
Figure 5

Figure 5. The interplay between the phosphorylation and SUMOylation of ETS-4.

A Effect of phosphorylation of ETS-4 on SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), HA-ETS-4(S73A) (SA), HA-ETS-4(S73E) (SE), T7-TDPT-1 and His-SUMO-2, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).

B Effect of SUMOylation of ETS-4 on PKA phosphorylation of ETS-4 in vitro. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(S73A) (SA) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation and then in vitro PKA kinase assays, using recombinant GST-fused active PKA, as indicated. Reaction mixtures were analyzed by immunoblotting (IB) with anti-phospho-PKA substrate, anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands. Asterisk indicates the non-SUMOylated- and phosphorylated-ETS-4 bands.
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 1

Figure 1 - Bak is present in the cytosol.

A Flow chart of human tissue homogenization and subsequent fractionation into cytosolic (C) and mitochondria-containing heavy membrane (HM) fraction. With authorization by the local ethics committee, human tissue samples were obtained from the Institute of Pathology, University Hospitals of Basel.

B Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control.

C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.

D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.

E Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 2

Figure 2 - Bcl-xL retrotranslocates Bak.

A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10.

B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.

C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.