EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Western blot analysis of immunoprecipitated Bur1-HA or Bur1-∆C-HA used in (B). (B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A beads, and then incubated with [γ-32P]ATP and GST, GST-N-Sch9 (1-390) or GST-C-Sch9 (391-824). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes exposed to x-ray film (top) or stained with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1. (C) Phosphorylation sites determined by mass spectrometry in Sch9 are indicated (red lettering). Green circle; Pkh target site (T570), blue circles; main TORC1 target sites (T723, S726, T737, S758, and S765). (D) Sch9-2D3E, but not Sch9-3A-2D3E, partially suppressed the rapamycin sensitivity of tor1∆ cells. tor1∆ which expresses pRS426 (mock), pRS425 TOR1, pVT102-Ura (mock), pVT102-Ura Sch9, pVT102-Ura Sch9-2D3E, or pVT102-Ura Sch9-3A-2D3E, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2-3 days. (E) The Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 3 days.
Fig.Figure 7LIN28A protein stability assays. HEK293 cells were transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G. Protein samples were prepared during 24 h of cycloheximide (50 μg/ml) treatment. Representative western blot data for LIN28A protein level changes normalized to β-actin protein levels (upper) or with normalization based on initial (0 h) LIN28A protein levels (lower). Band intensities were quantified using ImageJ, and normalized values are depicted (lower graph) (n=5). The proteasome inhibitor MG132 treatment increases LIN28A(R192G) protein level. After serum starvation for 3 hours, HEK293T cells expressing LIN28A(R912G) were treated with or without MG132 (0.1-10 uM) for 30 minutes, and then followed by WB analyses. LC-MS/MS analysis. Immunoprecipitated FLAG-LIN28A WT was loaded on an SDS gel (left upper). Bold red sequences indicate peptides matching LIN28A (left lower). Right, LC-MS/MS spectra of WT LIN28A. Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI. LET-7 microRNA regulatory activity of LIN28A. HeLa cells were transfected with plasmids expressing FLAG-tagged WT- LIN28A, LIN28AR192G, or mock pFLAG vector (control), and Let-7a, b, and c levels were quantified by RT-qPCR. The level of RNA was normalized to that of U6 snRNA. p<0.05, n=26 from 6 independent experiments, ns = not significant, ANOVA. Affinity of LIN28A protein binding to its cognate mRNA 3′UTR region. Upper: schematic for pmirGLO-LIN28A used in this assay, a bicistronic Firefly/Renilla luciferase (FLuc/RLuc) plasmid containing the part of the LIN28A 3′UTR containing the putative LIN28A protein binding sites (black dots)Wilbert et al., 2012(). Lower: HeLa cells were transiently co-transfected with pmirGLO-LIN28A 3′UTR along with pFLAG-WT-LIN28A, pFLAG-LIN28A R192G, or mock pFLAG control. LIN28A protein-mRNA binding activities were determined by relative luciferase activity levels of FLuc normalized to those of RLuc. *p<0.05, n=18 from 6 independent experiments. LIN28A-RHA co-IP assays in HEK293 cells transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G.
Figure 8Boxplot comparison of viral loads between severe-survival patients (yellow, n=8) and severe-fatal patients (purple, n=5). Line graph represents the temporal changes in viral load in time (days) after hospital admission of the patients. The cluster heatmap represents expression patterns of 1,541 proteins that exhibited temporal changes across time points in severe-survival (left) and severe-fatal (right) patients. The line plots indicate the expression trends of 1,541 selected proteins. VL, viral loads; FOT, the fraction of total. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range. Enriched annotations for corresponding clusters showed in Figure 6A (Fisher-exact test, p <0.05). (C, D) Systematic summary of proteins and signaling cascades significantly altered in severe-survival patients (lipid metabolism; C) and severe-fatal (viral life cycle; D). Values for each protein in all samples analyzed (columns) are color-coded based on the expression level, low (blue) and high (red) z-scored FOT. For each of the four panels, the heatmaps on the left indicate the Pearson correlation of proteins with viral load in severe survivors and severe fatalities, the heatmap on the right indicate the significant pathways enriched by proteins positively or negatively correlated with viral load in severe survivors and severe fatalities (Fisher-exact test, p <0.05).
Figure 9In non-cancerous cells (top panel), there is low level of Annexin A1 (ANXA1) and weak binding of FadA to E-cadherin (CDH1). In cancerous cells (bottom panel), Annexin A1 level increases, FadA binding enhances, FadA-E-cadherin-Annexin A1-β-catenin complex forms, β-catenin is activated, resulting in acceleration of cancer progression. CM, cell membrane.
Figure 9(A) The first instar TBPH-/- larvae were treated with Rapamycin for 72 h. Then the third instar larvae of W1118 and TBPH-/- were subjected to larval locomotion assays. Data from three independent experiments represented as means ± S.E.M., *, p<0.05; **, p<0.01, one-way ANOVA. (B) Similar experiments as in (A) were performed, but using PA instead of Rapamycin. The data from three independent experiments indicated the means ± S.E.M., **, p<0.01, one-way ANOVA. (C) The first instar TBPH-/- larvae were selected to standard food containing with Rapamycin and PA. Quantitative developmental viability analysis of W1118, TBPH-/- and TBPH-/- treated with Rapamycin and PA. The data from three independent experiments indicated the means ± S.E.M., **, p<0.01, one-way ANOVA. (D) A schematic model illustrates the molecular mechanism by which loss of TDP-43 enhances TFEB-mediated expression of ALP genes, blocks autophagosome-lysosome fusion, and induce neurodegeneration. TDP-43 directly targets raptor, a key component of mTORC1, to regulate mTORC1-TFEB signaling. Meanwhile, TDP-43 can affect the autophagosome-lysosome fusion in a Dynactin 1-dependent manner. The current study highlights the role of mTORC1 and Dynactin 1 dysfunction, altered autophagy and lysosomal biogenesis in TDP-43-mediated neurotoxicity.
Gene expression signature of the different cell-types in the zebrafish thyroid gland (A) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas.Figure 3(A) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas. The color scale represents expression as a z-score ranging from -2 (purple) to 2 (yellow). NFE: Non-Follicular Epithelium. (B - G) t-SNE plots overlaid with the expression of a gene specific to each of the cluster. The endothelium cluster (cluster #2) is a mix of blood vessels and lymphatic vessels while the immune cell cluster (cluster #4) is a mix of macrophages, neutrophils and lymphocytes The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (blue).
Figure 2H1299+shBRCA2DOX cells were grown for 4 days in the presence (-BRCA2) or absence (+BRCA2) of DOX and subsequently treated with 5 or 10 μM pyridostatin (PDS) for 3 days. Whole-cell extracts were immunoblotted as indicated. KAP1 and IRF3 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls. H1299+shBRCA2DOX cells were grown for 4 days in the presence or absence of DOX and subsequently treated with 10 μM pyridostatin (PDS) for 1, 2 or 3 days. Whole-cell extracts were immunoblotted as indicated. KAP1 and IRF3 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls. Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001. BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls. H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm. Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.
Figure 1A, B CB17-SCID male mice were injected intramuscularly into the hind leg muscles with (A) BRCA2-proficient (BRCA2+/+) or (B) BRCA2-deficient (BRCA2-/-) DLD1 cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 17, using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5. C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm. D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5. E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5. F BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red. G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.
SNX18 colocalizes with autophagic markers in the perinuclear area.Figure 3.(A) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting. Migration of different organelles is indicated, based on the positions in the gradient of calnexin (ER), GM130 (Golgi stack/CGN), SNX9 (plasma membrane), AP-1, TfR and Rab11a (Endosomes/TGN), and LC3-II and GABARAP-II (autophagosomes). (B) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130. Panels to the right show enlarged views of the boxed regions. (C) HeLa cells were transfected with GFP-Atg14L and immunostained for SNX18 and calnexin. The boxed regions are enlarged on the top and bottom. (D and E) HEK GFP-DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 (D) or WIPI2 (E). Results shown are mean ± SEM (error bars), n = 3 with 30 cells in each experiment. *, P < 0.05. (F) HeLa cells were transfected with control or SNX18 siRNA, starved for 2 h, and immunostained against SNX18 and Atg16L1. (G) HEK GFP-DFCP1 cells were transfected with control or SNX18 siRNA, starved for 50 min, and immunostained for Atg16L1. All images are from confocal microscopy. Inset panels show enlarged views of the boxed regions. Bars, 5 µm. See also Fig. S3.
Figure 1(A) Upper panels- Immunofluorescence imaging of PRR7 with Shank (synaptic) and MAP2 (dendritic) markers in DIV21 hippocampal neurons. Lower panels- Single plane apotome images of nuclei (DAPI) show PRR7 can be present (arrow) or absent (arrowhead) from nuclei. (B) Synaptic and dendritic distribution of PRR7 and pre (Bassoon) and postsynaptic (PSD95) markers (arrowheads-triple colocalization). (C) PRR7-GFP expressed in 17DIV hippocampal neurons is present in dendritic spines (arrows). Scale bar is 15 µm. (D) PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (Fibrillarin), SNAP25 and PSD95 were used as nuclear, presynaptic and postsynaptic markers, respectively.
Identification of RIPK2 PTMs during NOD signaling by mass spectrometry.Figure 2A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs. Protein lysates from FLAG-RIPK2 BMDMs (A) were sequentially subjected to ubiquitin enrichment (UBA, B) and FLAG pulldown (C) prior to protein elution and subsequent mass spectrometry analysis. B Schematic representation of RIPK2 PTMs detected in MDP-stimulated vs. unstimulated BMDMs. Red: phosphorylation, green: ubiquitination. N = 3 experiments C DiGly-modifications on RIPK2 in L18-MDP stimulated vs. unstimulated THP-1 cells determined by diGly proteomics. Shown are p-values and log2 differences of modified peptides based on imputed values; t-test. Score: peptide identification score as determined by MaxQuant. D Sequence conservation of stimulation-dependent modified serine (red) and lysine (green) residues amongst mammals. The degree of conservation is indicated by color saturation.
Figure 2(A) summ3-1 mutation in At2g11520. Exons are indicated with black boxes, UTRs with empty boxes, and introns with lines. (B) The predicted protein structure of SUMM3/CRCK3. The black box indicates the conserved kinase domain. The grey box indicates the signal peptide. TM stands for transmembrane domain. *, predicted MAP kinase phosphorylation sites. (C) Mutations in At2g11520 identified from 16 alleles of summ3.
Figure 7A) Immunoblot analysis. Primary hepatocytes were isolated from Nbr1f/f mice and then infected with adenovirus GFP or Cre for 48 hrs. Thereafter, the hepatocytes were divided into 3 groups: 1. Cultured in regular medium (-), 2. Treated with 10 μM As (III) for 12 hr [As (III)] and 3. After treated with 10 μM As (III) for 12 hr, cultured in regular medium for another 12 hr (Wash). Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nbr1f/f hepatocytes expressing GFP under normal culture conditions. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test. (C) Immunofluorescence microscopy. Nbr1f/f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and, when indicated, challenged with 10 μM As (III) for 12 hrs. The cells were then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. (D) Role of NBR1 in the p62-liquid droplets and the p62-mediated Nrf2 activation. Oxidative stress up-regulates the level of NBR1, which enhances the phosphorylation and liquid-droplet formation of p62. The resulting p62-droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2. Since p62 is one of the targets of Nrf2, increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress.
okp1-R164C restored binding of kinetochore components to centromeric sequences.Figure 3okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts. okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A). okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right) okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (-) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (-) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates.
a, Arrows indicate LAMP1– wild-type bacteria with actin ‘comet tails’ in cells infected for 6 h.figf4a, Arrows indicate LAMP1- wild-type bacteria with actin 'comet tails' in cells infected for 6 h. The boxed region is magnified in the bottom left panels. The merged image for this region is shown at the bottom right. Scale bar, 5 µm. b, Macrophages were infected with IPTG-induced iLLO bacteria. Arrowheads indicate actin- iLLO bacteria within LAMP1+ vacuoles. c, Macrophages were infected with wild-type (triangles) or IPTG-induced iLLO (circles) bacteria, and the percentage of LAMP1+ bacteria was quantified. Mean ± s.e.m. for three independent experiments. d, Macrophages were infected as in c with or without IPTG induction. Intracellular bacterial replication was determined using a gentamicin-protection assay. Shown is fold replication compared to 2 h post infection. Clear triangles, wild type; filled triangles, wild type + IPTG; clear circles, iLLO; filled circles, iLLO + IPTG. Mean ± s.e.m. or range for three (wild type, wild type + IPTG, iLLO + IPTG) or two (iLLO-IPTG) independent experiments, respectively. e, Wild-type or Atg5-/- MEFs were infected with iLLO bacteria, and intracellular bacterial replication was determined as in d. Mean ± s.e.m. for three independent experiments. Clear circles, wild-type MEFs; filled circles, wild-type MEFs + IPTG; clear squares, Atg5-/- MEFs; filled squares, Atg5-/- MEFs + IPTG. f, Wild-type (circles) or Atg5-/- (squares) MEFs were infected as in e with IPTG induction. The percentage of LAMP1+ bacteria was quantified as in c. Mean ± s.e.m. for three independent experiments. g
Figure 4Capsular invasion was defined as i) absent if tumor cells were separated from normal renal parenchyma by well-defined layers of FN or ii) present if strands of tumor cells were directly in contact with normal renal parenchyma Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on capsular invasion Incidence of Capsular invasion (stripes) in each model and treatment. 5-8 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. C) Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on tumor front of invasion Representative images of Vimentin staining (4X), as described in Figure 1, and box plot quantification of fold-invasion (∆ invasion) of treated vs control tumors indicating median, Q1/Q3 and max/min value whiskers. 6-12 animals per model and treatment group were analyzed by Mann-Whitney test where *p < 0.05, **p < 0.01. D) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion. Bar graphs show incidence of capsular invasion (stripes) in control and sunitinib treated tumors. 3-9 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. Box plots represent median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) of sunitinib-treated vs control tumors. 5-8 animals per model and treatment group were analyzed by Mann-Whitney test. *p < 0.05, **p < 0.01.
Figure 3Plasma level of metabolites (other than lipids) that are significantly different between Day 70 vs Day 0 in the CMA and placebo groups after weight loss adjustment are presented. A) Venn-diagram represents the number of significantly different metabolites (other than lipids) on Day 70 vs Day 0 in the CMA and placebo groups. Statistical significance is defined based on paired Student's t test. p< 0.05. 66 and 31 metabolites (other than lipids) were significantly different on Day 70 vs Day 0 in CMA and placebo groups, respectively. The two lists of metabolites had four overlaps. Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4); Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4); C) only in CMA (n=62) Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4); C) only in CMA (n=62) and D) only in placebo (n=27) groups are shown.
Figure 5(A,B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF, 30µg hTNF alone or in combination with 10000 IU mIFN-γ in wt C57BL/6 mice (A) or homozygous Flk1 dnIFN-γR1 transgenic mice (B). The line under the graph represents the treatment period. Tumor growth is depicted as mean TSI, error bars are SEM (n = 5). (C) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F (Y87F), hCD13-AFR or wt hTNF, alone or in combination with 200ng/ml hIFN-γ, measured via ELISA. (D) Schematic representation of AFN-II, consisting of a VHH fused via a 20xGGS linker to an engineered IFN-γ (e-IFN-γ) variant and N-terminal affinity tag. (E) Viability of B16Bl6 parental or mCD20-expressing cells after 72h stimulation with wt mIFN-γ or del8 mutant fused to a BcII10 or mCD20 VHH, in the presence of 60ng/ml mTNF. Cell viability was measured via an ATP luminescence assay.