EMBO/sd-character-level-ner huggingface dataset:
(B) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration. Two independent experiments were performed with three replications per experiment, each replicate consisting of three plants grown in individual pots (six measurements in total). The graph shows data points of the two individual experiments (I and II) along with their mean (Avg).
Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.
Sleep deprivation test of stx mutants. 12-hour mechanical night sleep deprivation was performed in stxd77 and w1118 control. Sleep amount calculation of deprived night sleep (Deprivation), the day sleep amount of previous day (Baseline) and the day sleep amount after the deprivation (Recovery) (For M, from left to right mean±SEM: 35.06±10.76, n=50; 370.8±16.07, n=50; 434.5±17.42, n=50; 59.36±11.59, n=53; 420.3±15.60, n=53; 523.9±15.12, n=53; For N, from left to right mean±SEM: 61.86±13.35, n=50; 104.0±11.86, n=53) Increased sleep amount in the Recovery sleep comparing to the Baseline which was calculated by the subtraction of the day sleep amount after the deprivation (Recovery) by the day sleep amount of previous day (Baseline).
(B) 35S labeling of de novo mitochondrial protein synthesis for 1 h, 72 h after transfection of HeLa cells with either a non-target dsRNA (NT) or LETM1 siR1; KBs (0.1 mM BHB or acetoacetate (AA)) replaced HG (25 mM glucose, 1 mM pyruvate) 9 hours before the labeling reaction. Polypeptide assignments flank the gel image; coomassie stained gels are used as loading controls, and immunoblots indicate the efficiency of LETM1 knockdown
I. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.
A Phospho‐peptide abundance during Cdc14‐induced mitotic exit. Examples of phospho‐peptides that disappear with early (red), intermediate (orange) and late (green) timing or that remain stable (black) are shown. This color coding is used throughout all panels of this figure.B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown, categorized according to timing of disappearance as described in (A).C Protein stability data for proteins corresponding to disappearing phospho‐peptides (blue) and for all other proteins (black).D The fractions of peptides that contain a minimal Cdk consensus motif ([S/T]‐P) within the timing categories shown in (B) were calculated. The ratio and P‐value of enrichment of each category relative to the combined stable/rest category (gray) are shown.E Similar to (D), the enrichment for reported physical Cdc14 interactors is assessed within [S/T]‐P‐containing phospho‐peptides.F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho‐peptides containing that motif.
C) Three Lamplight response categories - transient excitation (N = 35 biological replicates), transient inhibition (N = 6 biological replicates), and sustained inhibition (N = 8 biological replicates). Left panel shows representative single unit response to 2s 405nm or 525nm light. Mean baseline normalised firing (grey shading shows SEM) is shown in centre left (for entire recording, 500ms bins), centre right and far right panels (during 405nm and 525nm stimuli, respectively 100ms bins, N as above, except N = 5 biological replicates for sustained response to 525nm).
(A) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm.
(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.
A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication
Heatmap representing hierarchical cluster of 6716 DEGs (adjusted absolute log2(FC)>0.5, padj <0.05).
qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=5.
(C) EYFP expression in splenocytes isolated from uninfected (NI) vs MCMV-infected (MCMV) IFNbtm1(EYFP) reporter mice. The expression of indicated markers was analyzed on YFP+ cells. Plots are representative of one out of 6 mice from 2 independent experiments.
Tumor specific (human) enriched gene sets in Ren13 and Ren50 Ren-PDOX tumors, after GSEA analysis from RNAseq data. Gene sets most significantly enriched in Ren13 or Ren50 are indicated in the table (left), including their normalized enrichment score (NES), p-value and false discovery rate (FDR q-value). Heatmaps illustrate expression levels of the genes responsible for the enrichment of KRAS signaling up and down in Ren13 and Ren50, respectively (right).
(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.
B. Representative FISH images of cells treated with the indicated PMOs. Arrows point to peripheral RNA. Arrowheads point to perinuclear RNA. Boxed regions are magnified in the insets. Note that RAB13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region, while NET1 remains localized at protrusions. Scale bars: 10 μm (4 μm in insets).
(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.)
(B)Pull-down analysis for the hexameric IFT-B2 complex. A mixture of IFT172ΔC, IFT80, and the IFT57/38 complex ('input') shows only weak non-specific binding to GSH-resin ('empty'), but all proteins are pulled down by IFT54/20-GST complex. Note that the IFT20-GST and IFT38 proteins co-migrate on the gel and cannot be properly resolved. However, the presence of the IFT57 protein in the pull-down sample implies that its binding partner IFT38 is also there.
C qPCR of selected transcripts in SU-DHL-4 cells under conditions of non-targeting RNAi or hnRNP L knockdown with or without 50 μg/mL cycloheximide treatment (for 4 hours; n ≥ 3). Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO, and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO. Bars represent the mean ± SD. For clarity, only a single star was used to indicate significance; in all cases P ≤ 0.05 in two-tailed Student's t-tests.
(A) The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid second telogen phase. Images were captured at P60, P120 and P150.
G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections; blue=DAPI, green = CD68 (TAMs).
(B) Sequence identity between CSAG genes. Note isoforms of CSAG2/3 are splice variants.
A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection.
I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.
(A) Experimental scheme for generating BM macrophages. BM cells from Brd4fl/fl ERT2Cre mice were cultured in the presence of M-CSF for 7 days and treated with LPS. Brd4 was deleted by Tamoxifen treatment (see Figure 1B for verification of Brd4 deletion).
IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D.
male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole blood
A. Mitochondria visualized by MitoTracker (Red) when wild-type GASZ or GASZ with MLS mutation was expressed in HeLa cells. Scale bar: 15 m.
A, B, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test.
(A) Representative staining images (N = 5) of tissue from TNC high (WT/shC) and TNC low (KO/shTNC) tumors (N = 5 per condition) with the indicated antibodies. Arrows point at the respective immune subtype. Scale bar, 50 µm.
C. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVA
A, B Schematics of (A) gene editing strategy and donor DNA template for intron 1 CD40LG target locus and (B) experimental protocol in T cells.
Nucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.
SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown. Both control (CTRL) and DNMT1 siRNA-transfected cells were treated with 5-azadC.
(K-N) Example of a flow cytometry analysis (K and M) and final quantification of p-Akt (L) and p-Erk1/2 (N) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins in primed (blue bars) and restimulated (red bars) cells. In K and M, the grey shaded histograms represent the fluorescence obtained with the isotype-matched control antibody. In L and N, NSt, nonstimulated, St, stimulated. n = 3 per each experimental condition. Data information: values are shown as means ± SEM from three independent experiments. In panels L and N, P values are given relative to nonstimulated (blue asterisks) and stimulated (red asterisks) cells expressing Vav1WT. We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student's t test).
Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.
B RT-PCR for pro- and anti-inflammatory genes from microglia and culture supernatant levels of PGE2.
representative TEM images showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd).
A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b- positive Grn-/- microglia in comparison to wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed, are significantly changed, and from these 58 genes are up- and 58 genes are down regulated. Eight genes with the highest fold change are highlighted.
(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
BM macrophages (MQ) express HO-1. The MHCIIhigh MQ express higher levels of HO-1 than MHCIIlow MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO-1 in comparison to MQ.
(C, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).
D The distribution of nuclei per myotube were calculated on day 5 after differentiation. ** p < 0.01; * p < 0.05; one-way ANOVA followed by Bonferroni post-hoc test.
A. Pharmacological (INK-128) and physiological (glutamine deprivation) inhibition of the mTOR signaling.
High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.
C Schematic diagram showing potential fates of cellular pyruvate. PDH, pyruvate dehydrogenase; CS, citrate synthase; GLS, glutaminase; PC, pyruvate carboxylase; GOT2, glutamic-oxaloacetic transaminase 2; GPT2, glutamic-pyruvic transaminase 2.
A Closeup of the central symmetric dimer of the AMP-PNP particle from the membrane distal view. Interfaces within the symmetric dimer are formed intramolecularly between the FERM and kinase domains (F and K; F' and K'). Intermolecular interfaces are formed within the symmetric dimer between F and K', F' and K as well as K and K'. Intermolecular interactions that link symmetric dimers are formed between 2 FERM domains (F and F*; F' and F**). Coloring is as in Fig.1E. The FERM subdomains F1 and F3 (F2 is not visible from this view), kinase N- and C-lobes (KN, KC), AMP-PNP and the N- and C-termini (N, C) are labelled.
RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.
A: Description of the experimental strategy. LA liposomes (endosome-like) are decorated with cSTD3 or pS209 cSTD3 owing to covalent links with MPB-PE (1.2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide]), and mixed with LB liposomes (ER-like) covered by VAPHIS6 attached to DOGS-NTA-Ni2+ (1.2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl], nickel salt). For dehydroergosterol (DHE) transport experiment, LB liposomes also contain DHE and a dansyl phosphatidylethanolamine (DNS-PE). The transport of DHE from LB to LA liposomes is followed by measuring the change in FRET from DHE to DNS-PE.
E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.
qPCR analysis of miR-483-3p/-5p in serum from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups
(G) Scheme of TMEM59 showing amino‐acid positions and the minimal active subdomain (amino acids 263-281). ED, extracellular domain; TM, transmembrane domain.Source data for this figure is available on the online supplementary information page.
1.SARS-CoV-2 activates delayed innate immune responses in lung epithelial cells Measurements of replication and innate immune induction in Calu-3 lung epithelial cells infected with SARS-CoV-2 at MOIs 0.08, 0.4 and 2 TCID50VERO/cell. (A) Replication of SARS-CoV-2 genomic and subgenomic E RNAs (qRT-PCR). (B) Quantification of N staining from cells in (A) by flow cytometry. Mean percentage of N-positive of all live-gated cells is shown +/- SEM, n=2.
d. Pseudotime analysis of intrathecal B cell subsets in inflammatory pseudotumor CSF with the naïve B cell cluster, identified by canonical markers, as root node. Percentage of cycling naïve B cells and IgG4 B cells depicted in pie charts.
F. Jurkat cells were transfected with DRP1S637A-GFP, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.
(e) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control, HDAd-TFEB and Tcfeb-LiKO (liver KO) mice treated as indicated. Bar graphs show mean ± s.d. for n = 4 mice. *P≤0.05; **P≤0.01.
d, LC3 translocation to the phagosome is induced by TLR signalling. GFP-LC3 was transiently transfected into wild-type or Tlr2-/- macrophages, which were exposed to zymosan for 45 min.
Representative confocal microscopy images of F-actin and 3D reconstruction of z-stacks from nuclear region with LifeAct-mCherry Venus expressing cells with treatment as indicated (B) (n = 3 independent experiments). Red: LifeAct, Blue: DAPI. shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.
(B) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V. parahaemolyticus. Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains. Outputs represent eluates from protein A/G beads cross-linked with anti-EBP2 (IP: anti-EBP2) or anti-VgpA (IP: anti-VgpA) antibodies and analyzed with anti-VgpA and anti-EBP2 antibodies.
A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.
Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) assessed at around four weeks post tumor cell injection.
(C) The degradation of 3HA-Pgc1 was analyzed as in (A) in cells bearing the CDC48 temperature-sensitive allele cdc48-6. Cells were grown 2,5 hours at 37C prior addition of cycloheximide. Inactivation of Cdc48 mutant protein was confirmed by stabilization of the ERAD substrate Erg1 in the same cells.
(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HeLa (m-p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.).
B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).
Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B).
(E) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter. 3 (1,25D3)‐ (F) and retinoic acid (RA)‐regulated (G) luciferase expression is dependent on the ER8 element in the p19ink4d promoter. Luciferase expression driven by a thymidine kinase (tk) control promoter is shown.
B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line. Data are presented as mean ± SEM (n = 4). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0066 (S6 naïve vs S6 APOPT1GFP), ***P = 0.0006 (S6 EV vs S6 APOPT1GFP), *P = 0.0169 (S2 naïve vs S2 APOPT1GFP), ***P = 0.0008 (S2 EV vs S2 APOPT1GFP)
H Representative immunofluorescent images of T40PL cells transfected with TFEB and triple staining for TFEB (FLAG), PTEN and PHF1. Arrow marks cell with higher TFEB and PTEN is correlated with lower PHF1. Arrowhead indicates nearby cell with opposite TFEB/PTEN/PHF1 patterns. Scale bar: 10 μm. Each bar represents average ± s.e.m.
B. Superposition of the genuine mouse and the human chimeric β5c/β6 active sites according to panel A proves the proper orientation of Met45 in the 'closed' conformation.
J. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were digested with AflIII and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated.