EMBO/sd-character-level-ner huggingface dataset:
E. Tumor size in individual mice from Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.
(f) The heatmaps show z-scores for the individual marker genes of the indicated cell types across IPF stages and controls.
(B) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001).
(G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.
(E) IL-6 protein released from mock or TLR2 Cas9-CRISPR-edited THP-1 cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD) ), Student's t-tes
(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.
(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).
(G) Schematic illustration for the experiments shown in H, I. (H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm. (I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).
H Co‐IP of wild‐type or PMB‐mutated LAPTM4B with wild‐type or kinase dead PIPKIγi5 in HEK293 cells.
B. Immunofluorescence on meiotic chromosomes from WT and Brca1ex2/ex2;Trp53bp1-/-spermatocytes. Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body. Scale bar: 5 apsi
(B) Sedimentation profiles of ERAL1, MRPS28, EFG1, CLPP, CLPX, EFTU and MTIF2 in heartmitochondria as in A.
Growth analysis of wild type Xcc8004, EPS deficient mutants (B5 and B12), ndvB and ndvB (pHMJ) grown in PS medium (A); PS medium containing 50 µm extracellularion chelator, diethylenetriaminepentaacetic acid (DETAPAC) (B); or supplemented with either with (C) secreted glucan (0.5 mg/ml); (D) xanthan (0.5 mg/ml); (E) FeSO4 (40µM); and (F) MnCl2 (40µM). Data shown are mean ± SD (n=3). From here onward, until not mentioned, the 'n' indicates three biological replicates.
B. qRT-PCR analysis validates that mRNA of MBNL1-3, SART3, RSRC1 and other splicing factors are de-repressed by miR-10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression.
d, Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes.
(F-G) Intracellular cytokine staining (F) and percentage of IL-22+ILC3s (G) after stimulation with αCD1d, IL-23 and/or IL-1β. *p<0.05, **p<0.01 two-tailed unpaired t-test (n=4-8)
D A multinucleated cell isolated from a Stx2repro34 mouse testis, showing four nuclei. Differential interference contrast (left) and Hoechst-stained (right) images. Bar = 10 μm.
Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).
E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).
F Samples of a patient with M. xenopi osteomyelitis from repeated biopsies at onset of disease (1st) and 2 months after (2nd) were quantified by immunohistochemistry for p53 and hematoxylin counterstaining.
B. MCU silencing blunts cell invasiveness. Stable shMCU and shControl-expressing spheroids were plated and let grow into collagen-I (0 hour time-point). Spheroid area was measured at 0-hour and 48-hour (n=8). Scale bar 300 μm. P-value: ***p=0.0003.
(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.
F) tlc1 cells reach senescence at PD 80-100. %viability of indicated strains after propagation in YPD is indicated at different population doublings n=3. Asterisk indicates population doubling used in G) to determine binding of Npl3 to short telomeres by TAP-ChIP. 3 independent spores from the indicated genotypes were used. Population doublings are counted from germination of meiotic products and hence begin at approx. PD40. Abbreviations: TAP: tandem affinity purification tag
F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f
(D, E) Endocytosis (D) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).
F- Alternative splicing events [in %] detected in 335 differentially expressed isoforms upon CTD shortening in U2OS cells.
F. PEBP1 knockout enhanced HIV-1 transcription in the primary CD4+ T cell model of latency. The expression of HIV-1 was measured by nanoluc after gene knockout where α-CD3/CD28 stimulation served as a positive control.
(a, b) The heat maps show the deconvolution scores of cell types across BALF samples grouped by diagnosis (a) and indicated clinical parameters (b).
(m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.
(H, I) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. DCs (H) and LCs (I) were exposed to either the untreated or pre-treated SARS-CoV-2 isolate (100 TCID/mL) for 24h, washed thoroughly and co-cultured with Huh7.5 cells. Quantification of viral RNA was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. (H, I) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (H) (n=3 in duplicates), (I) (n=4 in duplicates). DCs: Dendritic cells, LCs: Langerhans cells
The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV A. Mean ± SEM 24(S)-hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt (n=6 mice, 14 week-old and 16 week-old, 2-way ANOVA, **p6 weeks = 0.0023) and NPC1nmf164 (n=5 mice, 14 week-old and 16 week-old, 2-way ANOVA, ***p6 weeks < 0.0001; ***p8 weeks < 0.0001) mice.
(B) Schematic depiction of miRNA precursors. Precursor sequences and predicted basepairing were adopted from miRbase miRNA annotations miR-10b secondary structures in the mirRbase contained two different basepairing versions in the loop, the alternative folding is displayed
RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.
E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.
Cartoon illustrating the recognition of Nek2A by APC/CMCC. KEN box and D box dependent substrates such as securin and cyclin B do bind APC/CMCC. In contrast, cyclin A and Nek2A bind APC/CMCC-open. Cyclin A is ubiquitinated by UbcH10 and Nek2A has more chances of being ubiquitinated once bound to the APC/CMCC-open by using both UbcH10 and UbcH5. The positioning of UbcH5 with respect to the APC/C catalytic site is based on mutagenesis data
H Ratio of enrichment of total histone H3 to acetylated H3 (AcH3) in Jurkat T cells at the indicated target promoters quantified by ChIP-qPCR. Data are presented as mean of % input (non-IP control) ± SD of three biological replicates.
Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.
(c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated.
(C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.
B. Analysis of detergent resistant recombinant FadA polymers expressed in E. coli. An aliquot of 50 μg detergent-resistant pellets prepared from E. coli BL21(DE3) carrying the cloning vector pET21(b), pYWH417-6 (p_FadAc), or pYWH418 (p_mFadA) were analyzed by SDD-AGE, followed by Western blot analysis. FadA polymers were only detected in E. coli expressing FadAc, but not in mFadA or the vector control.
Representative staining of nestin and periostin in femur sections from 8-day-old wild-type and Dnmt2−/− mice. The anti-periostin staining is comparable in the periosteum of wild-type and Dnmt2−/− mice, but is reduced in the bone marrow cells (compare the highlighted areas). Scale bar: 100 μm. Nuclei are counterstained with DAPI (blue).
A Test procedure of NORT (left panel) and the bar graph showing the exploratory preference for novel object of offspring mice (right panel). F, familiar object; N, novel object.
F-H. Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in (D). Data (F) are representative FACS plots, gated on VD-CD90+CD4+CD44+cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group. (G) Mean fluorescent intensity (mean +SEM) of IL-17A staining by MOG-specific Th17 cells, shown in (F, upper row). (H) Quantification of MOG-specific GM-CSF+ Th17 cells (mean +SEM) shown in (F, lower row).
Comparison of the distribution of ChIP-seq reads across the indicated categories of genes for Sen1 (this study) and Seb1 (data from (Lemay et al, 2016)).
(l) Diffusion map of mouse AT2 cells colored by cell type identity and inferred pseudotime.
ChIP assays detecting binding of LRP130/PGC1α to putative binding sites in the FOXO4 promoter using qPCR (top). Data were normalized to the IgG negative control. Schematic illustration of the LRP130/PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter (bottom).
(a) Ba/F3 cells expressing myrPI(3) K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3) K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate.
(H) ChIP-seq analysis of DAXXMycHis occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) versus GAL4DBDCBX2.1 and DAXXMycHis (Y-axis) showing strong enrichment (log2) for DAXXMycHis at many TSSs of genes, but only when both proteins are expressed (Y-axis), indicating that CBX2.1 targets DAXX to chromatin. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.
RT-PCR analysis of expression of Mmp9 in murine Cdkn2a-/-;p53-/- and in WT placentas on day E14.5 (n>=4 fields of view for each genotype). Values are means + SEM of at least three experimental repeats. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.
E BGL3 deficiency inhibits DNA damage repair. Quantification of γ-H2AX foci at indicated times after irradiation (2Gy) are presented. Data shown are results of three independent experiments (100 cells for each experiment), presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.
(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).
C Graph showing in vivo growth of the different cell line xenografts. Curves represent mean log-transformed luminescence ± SEM of individual glands; MCF7 (n=17), T47D (n=10), MM134 (n=12), and SUM44 (n=12). Statistical testing for interactions group cell growth/ time relative to MCF-7 was carried out by applying mixed-effect linear models. P-values are associated to likelihood-ratio test statistics of the ANOVA function in R.
(c) FACS-sorted HE cells were subcultured with TCP (300 nM), UK5099 (10 µM) or both and day 3 CD43+GPA+ cell frequencies ± SEM relative to the control are shown (n=4 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.
C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows respiratory control rate (RCR) calculated as described in materials and methods. Graphs represent mean± SEM. N=4-5, ANOVA followed by Newman Keuls test
(A, Human (A) lung sections were stained with H&E or with Periodic Acid Schiff (PAS) and Alcian blue (AB). Scale bar, 50 μm.
(F) A model of the molecular basis of accessible PM cholesterol recognition by the GRAM domain of GRAMD1b. At steady state, limited amount of accessible cholesterol (i.e. chemically active cholesterol) in the inner leaflet of the PM is not sufficient for the recruitment of the GRAM domain to the PM. Upon additional cholesterol loading to the PM or liberation of cholesterol from sphingomyelin (SM)-sequestered pool of inaccessible PM cholesterol (i.e. chemically inactive cholesterol), accessible PM cholesterol transiently increases in the inner leaflet of the PM beyond a certain threshold (as unsequestered cholesterol can spontaneously flip flop between the outer and inner leaflets), resulting in increased co-distribution of accessible cholesterol and anionic lipids, including phosphatidylserine (PS), in this leaflet, which serves as a platform for PM recruitment of the GRAM domain. The GRAM domain then binds to both accessible cholesterol and anionic lipids, using distinct but synergistic binding sites for cholesterol and anionic lipids, to tether the PM to the ER, and facilitates the StART-like domain-dependent extraction and transport of accessible PM cholesterol to the ER. Accessible cholesterol transport to the ER results in suppression of SREBP-2 cleavage, preventing overaccumulation of cholesterol in cells. Our study identified amino acid residues that are important for cholesterol sensing (G187, R189) and anionic lipid sensing (K161, R191) of the GRAM domain. G187 is critical for determining the sensitivity of the GRAM domain to accessible PM cholesterol, while K161 and R191 contribute to a basic patch of the GRAM domain that is required for sensing anionic lipids, including PS. An intellectual disability-associated R189W mutation, which specifically results in cholesterol-sensing defect, impairs the GRAM domain's ability to sense transient expansion of the accessible pool of PM cholesterol and abolishes GRAMD1b-mediated PM to ER cholesterol transport.
B Representative confocal images of BrdU-incorporating cells (magenta) assigned to distinct cell types of 8 week-, 14 month-, and 21 month-old hGFAPeGFP mice: rNSCs [identified by their SOX2+ (green) cell bodies residing in the SGZ with a GFAP+ radial process (cyan)]; IPCs/neuroblasts [SOX2+ (green) nuclei located to the SGZ, DCX+ cells (white) in the SGZ and GZ, respectively], and astrocytes [SOX2+ cells (green) in the hilus, GZ, and ML]. Data information: ; dotted lines border SGZ and GZ; scale bars = 50 µm.
A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.
Detailed view of the primary and secondary interfaces observed in the EschOTU-Ub structure. EschOTU (orange) and Ub (red) residues participating in the interface are shown with ball and stick representation.
(D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.
Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV-1 was evaluated 72 h post-infection by luciferase activity
A. Remazol brilliant blue assay. The absorbance of supernatants from the reaction containing purified and RBB-labelled E. coli peptidoglycan and the indicated protein (50 µg) were measured at =595 nm after incubation for 0.5 or 4 hours at 37°C. The results shown are the average and standard deviation from triplicate reactions (n=3). Asterisks indicate significant differences compared to the buffer (NS, non significant; **, p < 0.01; ***, p < 0.001). The supernatant of the reaction after 4 hours of incubation is shown on bottom.
(A) Haematoxilin and Eosin staining of muscle biopsies from Pt 01, Pt 02 and Pt 03 performed 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei.
Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).
CD68 immunoreactivity in white adipose tissue (scale bar = 25 μm, images representative of one animal each from a total of four animals per experimental group). Arrow points to a region stained with CD68 antibody. *P = 0.0109, Student's t-test.
The relative expression of TERC was measured by RT-qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used as internal control (n = 3 experimental replicates). qPCR of 3'-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n=3 experimental replicates). qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n=3 experimental replicates).
(G) Quantification of the fraction of growing (>50 μm) axons in one imaging field (WT: control 76.8±5.7%, Sema3A 57.7±10.6%, p<0.001, crmp2-/-: control 75.4±10.4%, Sema3A 75.4±6.7%, p=0.99), n=3 experiments per genotype, 7-8 explants per condition. Mean ± SD. *** p<0.001, 2-way ANOVA with Bonferroni's multiple comparisons test.
(F) MA-plot showing FC (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.
(D, E) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores.
(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).
(c) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.
(A) Volcano plot of proteomic data reveals significant changes in proteins of the respiratory chain and factors involved in mitochondrial protein synthesis (FDR < 0.05).
B-D. The in vitro synthesized radioactive single or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria, oxidized, and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.
E DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).
E, Fluc-EGFP specific luciferase activity measured in the indicated brain regions of Fluc-EGFP mice at the indicated ages. Values are normalized to the 3-month-old group. N=5 Fluc-EGFP mice for each age group. One-way ANOVA with Bonferroni's multiple comparisons test. ANOVA: Hippocampus, *p = 0.0205; Cortex, **p = 0.0077; Cerebellum, n.s. Significant pairwise comparisons to the 3-month-old group are indicated on the graphs.
Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).
(I) HEK293T cells transfected with GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to immunoblotting with anti-GST, anti-Strep, or anti-β-actin antibody.