EMBO/sd-character-level-ner huggingface dataset:
B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.
A FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24-/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± s.d. of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab.
Representative images of pERK1/2-stained lung sections from the indicated genotypes. Scale bars, 100μm. Quantification of pERK1/2-positive cells/high power field (HPF) in lungs of the indicated genotypes (n = 6 per genotype).
Endogenous SOX2 levels in single cells (turquoise) normalized to the value at t=0 in SNSF cells upon induction of SOX2-SNAP (n=37, left) or a truncated SOX2 version missing the DNA binding domain (YPET-SOX2-delDBD, n=47, right). Black line: average SOX2-SNAP levels after induction normalized to the maximum level (n=22); Yellow line: average YPET-SOX2-delDBD levels after induction, normalized to the maximum level (n=20).
(f) Left: confocal images of LC3-positive dots. N, nucleus. Scale bar, 10 μm. Right: percentage of cells containing more than five LC3-positive dots. At least 60 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA.
e, Reconstitution of ARF1-HisN-myristoylation (N-myr) by NMTp in bacterial cells and IpaJ cleavage reaction in vitro (see Supplementary Information and Methods).
Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.
(E) Cryo-EM density map of the NHE9 ∆CTD homodimer with the 6-TM core transport domains (coloured in blue), the dimer domain (coloured in orange), and the linker helix (coloured in grey).
Mice were fed chow or the HFD for 9-10 weeks then treated with vehicle or 120 mg/kg SH-BC-893 Mondays, Wednesdays, and Fridays for 3 weeks; n=9. Mean absolute (O) or relative (P) caloric intake per mouse in the 24 h after treatment is plotted for each of 8 different treatments; mice were euthanized 4 h after the ninth treatment.
Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.
Most uORFs are not conserved at the peptide sequence level - Pie charts depict coding potential (phyloCSF score) of (A) all potential uORFs and (B) translated uORFs. uORFs with a phyloCSF score ≥ 50 were considered conserved, uORFs were considered weakly conserved if their phyloCSF score was positive but less than the conservation threshold of 50.
H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice.
Mapping of the single cell data shows different distribution of the respective clusters. Astrocyte clusters 0 - 4, 9 and 10 are shown in panels A - E and J, K, respectively. Data information: Color-coding bars represent degree of gene expression similarity between all cells from given cluster and mixture of all cells from given single pixel of Slide-seq data. Scale bars: 300 µm
H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.
B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.
A, B. HeLa cells (A) or SH-SY5Y neuroblastoma cells (B) treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell (A, HeLa, Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; **** P ≤ 0.0001; N (cells from 3 independent experiments) = Ctrl/EV: 81; Ctrl/EV/Torin1: 48; Ctrl/C9orf72L: 86; Ctrl/C9orf72S: 48; Rab1a/EV: 79; Rab1a/EV/Torin1: 68; Rab1a/C9orf72L: 78; Rab1a/C9orf72S: 52; B, SH-SY5Y: Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; ** P ≤ 0.01; **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 70; Ctrl/EV/Torin1: 56; Ctrl/C9orf72L: 45; Ctrl/C9orf72S: 43; Rab1a/EV: 63; Rab1a/EV/Torin1: 55; Rab1a/C9orf72L: 44; Rab1a/C9orf72S: 37). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.
TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments.
(A) Western blot analysis of CTF beta (CTFβ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti-APP-CTF antibody Arrows point to phospho-C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH. (B) Quantification (mean ± SEM) of phospho-C99/C99 CTFβ species, normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs, 5xFAD/KO; ***, P<0.001 5xFAD vs. 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P=0.056 5xFAD/ΔDD vs. 5xFAD/KO. N=5 mice per group.
C. U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC.
B-D Schematic representation and structure of semi-open ABCE1 as in 80S-pre-splitting complexes (Brown et al., 2015, PDB 3JAH) (B), hybrid semi-open/closed ABCE1 as in native 40S initiation complexes (C) and fully closed ABCE1 as in in vitro reconstituted post-splitting complexes (Nürenberg-Goloub et al., 2020, PDB 6TMF) (D). Nucleotide binding sites colored in blue and bound nucleotide indicated with yellow circles (one circle per phosphate group).
A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.
(A) Design of dynamin mutants to restrict the PHD in the closed (Dyn1Closed) or open (Dyn1ΔΔ) state.
C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.
(C) Cancer tissue sections were processed for IHC using antibodies against endogenous GOLPH3 and LCS. The IHC intensity of the staining for each section and for both the tested proteins was classified according to the criteria described in Materials and Methods.
Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.
Schematic illustration of the strategy to apply single-cell RNA sequencing (scRNAseq) on FACS-collected homogenous Hb9:GFPon MNs.
A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666
H. GSEA indicating significant enrichment of genes of the JAK/STAT signaling pathway of the KEGG pathway database in the DN2-like M2 AML samples as compared to the residual M2 AML samples using the Verhaak AML data set (Verhaak et al., 2009).
(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.
I: ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC-PDX tumors compared to the control tumors. 18S gene was used as internal control. n=5 animal per group and 3 technical replicates presented as the mean ± SD
A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1).
cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Co-isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ-irradiated HEK293 cells expressing GFP-hcGAS or GFP-hcGASΔDNA probed for indicated proteins.
D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD).
(A and B) Volcano plots showing the relative abundances of transcripts in WT and MZsinhcaf -/- FG follicles (A) or mature eggs (B). Transcripts were differentially expressed if p< 0.05 and fold-change >2 in MZsinhcaf -/- FG follicles or mature eggs compared to WT.
(H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.
C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.
Line chart of 50 representative highly expressed female genes The dot is the mean value of FPKM in three repeats.
(a) 264 common genes were identified down-regulated by both deficiency for YAP/TAZ and deficiency for ATF4, among which were known ATF4 targets, such as SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siYAP/TAZ or siATF4 and cultured with 6μM Sorafenib for 18 hours and RNA was subjected to next generation RNA sequencing. Venn diagram analysis was conducted using VENNY 2.1.0.
F. Relative FCR activities in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.
Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).
B Electrostatic surface representation of the structure (back view from A). The bundle region of TMs forms a tunnel that binds monoolein, and their splitting generates a lateral opening inside the membrane.
(a) Non-fasting blood glucose level was monitored weekly in Atg7+/+, Atg7+/−, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice. ***P0.001; two-way ANOVA.
E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.
Proportional changes in Her6::Venus(+) cell numbers during development; bars indicate median and interquartile range of counts collected from 3 different z-stacks per embryo and one embryo per condition; one-way ANOVA with Bonferroni multiple comparison test with significance p<0.01
(b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student's t-test.
A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-mybexpression for each condition.
(J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3
B Immunohistochemistry detection of NFATc1 and p53, illustrating G1‐G2 and G3 in human PDAC tissues. Scale bars, 100 μm.
(b) Binding of NSP2 to a fluorescently labelled 20-mer ssRNA in the presence of 4.7% propylene glycol (PG), measured by fluorescence anisotropy. Data points represent mean anisotropy values±SD from three measurements (technical replicates, values normalized from 0 to 1).
(b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.
(B) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA-resistant HA-GRWD1 were treated and analyzed as above.
(H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.
(A) UMAP visualisation of the B cell/B-ALL compartment. From left to right panel, cells are coloured according to: unsupervised clustering (Louvain - resolution 0.6), experimental condition and marker-based custom cell-type classification, unequivocally identifying leukemic cells by the presence of the tTA transcript.
C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.
(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).
B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.
A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (B) RT-qPCR analyses
(A) A scheme showing experimental timeline when neuronal inhibitors were added to and removed from the cultured neurons. A cocktail of TTX, D-AP5 and NBQX was added to neurons at DIV 21.
D. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control.
Fluorescence t 1/2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS‐Swi6‐EGFP and NLS‐Swi6‐KR25A‐EGFP that reside in the G2 of the cell cycle.
(A) Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ratios of ATAC-seq signal for the indicated group of peaks, comparing the indicated genotypes or treatments. Core promoter peaks are <300 nt from a TSS; Extended promoter are more than 300 nt but <2 Kb for a TSS; Gene body peaks overlap transcription units, and Intergenic do not. Each group of peaks was compared to the intergenic peaks. In addition, for each group of peaks, the peaks within 1 Mb of Reno1 where compared to peaks further away. P-values computed using two-sided Wilcoxon rank sum test, and shown only for cases where P<0.05. Number of data points (from left to right): 39; 10,777; 11; 3,550; 17; 14,380; 18; 20,930. (B) Changes in ATAC-seq signal for peaks assigned as falling within core or extended promoters, of genes in the indicated group (up/down-regulated by at least 30% and with adjusted P<0.05) in cells with Reno1 (left) or Bahcc1 (right) KD. P-values computed using two sided Wilcoxon rank sum test.
DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.
Surface representation and cross section of Tle1 from P. aeruginosa (PDB 4O5P) (A) and EAEC - this study (B). The active site is shown in red. The substrate entry channel is indicated when present.
(b) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP-PLC(PH) together with empty vector (control) or dominant-negative dynamin II mutant (K44A dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note the co-localization of Atg16L1 with PLC(PH) at the plasma membrane; this is quantified in the graph at the right. Asterisk, P 0.01. n = 20 cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole.
(B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison.
D Accelerating rotarod was assessed after 7 weeks of treatment. **P < 0.01 and ***P < 0.001 compared to vehicle-treated wild-type (WT) mice; #P < 0.05 compared to vehicle-treated R6/1 mice. WT veh N = 13; R6/1 veh N = 9; R6/1+BA N = 9.
A Relative mRNA levels of the macrophage marker F4/80 in SVCs isolated from epididymal adipose tissue. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0279 (Student's t-test).
(e) Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa cells. Error bars show S.E.M from 3 experiments, *** = p<0.001.
(D) p53-/- hRasG12V MEF were treated with DMSO or 1µM SNS-032 (SNS) for 1h. Nuclear lysates were analysed by Western blotting
G. Working model: LORE is phosphorylated at tyrosine600 (Y600) at a basal level. In perception of 3-OH-C10:0, LORE Y600 phosphorylation is elevated, which then trans-phosphorylates PBL34 and likely its paralogs. Phosphorylated PBL34 dissociates from LORE, leading to immune activation. In this event, PBL34 kinase activity is required. However, the bacterial effector HopAO1 targets LORE Y600 and dephosphorylate the tyrosine-phosphorylated Y600, which subsequently suppresses immune response in Arabidopsis.
(c) We transfected MN-1 AR65Q cells with the mCherry-EGFP-LC3 construct and with either BFP-empty or BFP-TFEB. Autophagic vesicles forming in BFP-expressing cells were then detected as red, green or yellow puncta, as shown. Scale bar, 20 μm. (d) Left, quantification of autophagic vesicle type. Note marked reduction in autophagosomes in MN-1 AR65Q cells expressing BFP-TFEB; n = 3 independent experiments, F = 16.596, ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. Right, calculation of the autophagy index; n = 3 independent experiments, F = 24.43, ANOVA with post hoc Tukey test. **P 0.01. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
mucosal hemorrhages (G; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. ns: not significant.
RT-PCR P120 reporter assay(Hall & Padgett, 1996) to measure the relative usage of A-type (AT-AC) and G-type (GT-AG) 5' splice sites in direct competition. Upper: schematic diagram showing the overall architecture of the reporter construct with its down- and upstream splice site (thick red and blue bars, respectively) and the products created by splicing (Dn and Up, respectively). Lower: RT-PCRs of the reporter with A- or G-type splice sites in the down- or upstream positions as indicated below the gel. SS, splice site. *, PCR product after use of a cryptic major splice site (not shown in the schematic).
(f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody.