EMBO/sd-character-level-ner huggingface dataset:
Figure 7BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue). BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue). Glycosylation of the IL-2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag-tagged WT IL-2Rα protein was expressed in HeLa cells with (+) or without (-) BAG6 siRNA, and was immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F, and subjected to Western blot analysis with an anti-Flag antibody. Low-mobility (indicated as glycosylated) and high mobility (indicated as non-glycosylated) signals of WT IL-2Rα are indicated. Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Representative image from a cell surface glycoprotein quantification assay with Alexa FluorTM488-conjugated Lectin GS-II as a probe (D). Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E). Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).
CHK1-deficiency causes stem cell loss in the fetal liver.Figure 5Representative dot-plots of the LSK cell phenotype of Chk1fl/+ and Chk1fl/- Vav-Cre embryos observed in E13.5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells, as well as LKlow and LSKlow cells, found in the fetal liver on E13.5. Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre. Colony formation potential of FACS-sorted LSK cells (left panel, N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre) and total fetal liver cells (right panel, N=3 for Chk1fl/+ and N=6 for Chk1fl/- Vav-Cre) in methyl-cellulose assays. Bars represent means ± S.E.M. Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 (N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre), to discriminate LT- and ST-HSC from MPP within the LSK-subset. Bars represent means ± S.E.M.
Treatment with 24F4A leads to a dose-dependent downmodulation of CD32a on pDCs.Figure 5A-F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.
Figure 1(A,B) Representative B16F10 tumor established in a syngeneic mouse showing that tumor vessels, identified by CD31 immunostaining, are generally p-EphrinB+ (scale bars: 500μm in A; 50μm in B). (C) CD31+ endothelial cells lining a vessel are mostly p-EphrinB+ whereas NG2+ perivascular cells are mostly p-EphrinB-; cell nuclei are identified by DAPI; confocal images (scale bar: 10μm). (D) EphrinB2 is detected in cell lysates of 4T1, LLC1 and HUVEC, but not in B16F10 cells; representative of 3 immunoblotting. (E) EphB4 depletion in B16F10 cells; immunoprecipitation/immunoblotting; control: no infection; pLKO: infection with control lentivirus; shEphB4: infection with silencing lentivirus. (F) B16F10 tumors induced by inoculation of EphB4-depleted B16F10 cells contain reduced levels of EphB4 compared to control pLKO-infected B16F10; representative immunoblotting (of 16 mice); TRP2: tyrosine-related protein 2 (TRP2). (G) CD31+ vessels in EphB4-depleted tumors are generally p-EphrinB+; scale bar: 200μm. (H) Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC; immunoblotting results; NA3VO4: sodium orthovanadate. (I) Tumor vessels in B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+; confocal images (scale bars: 200μm).
Figure 7Upon callus induction, HAG1 mediates epigenetic reprogramming of WOX5/WOX14, SCR, and PLT1/PLT2 by catalyzing histone H3 acetylation at their chromatin. This epigenetic control on CIM assures the proper induction of these root transcription factors that act as potency factors establishing competence to regenerate shoots on SIM.
Figure 4A. Primary hippocampal neurons were treated with a mixture of 3-miR mimic or control oligonucleotides and follow up analyses (imaging, electrical recordings) were performed. B. Functional mature synapses were quantified via co-localizations of pre- (Synatophysin 1) and the post-synaptic (PSD-95) markers and compared between 3-miR-mix and control groups. Scale bar: 10 μm. Two independent methods (SynQuant and Colocalization) were used for quantification. 3-miR-mix reduced the number of functional synapses compared to controls (n = 24-30 images) C. Dendrite labeling and quantification. Dendritic spines were stained with Dil. Scale bar: 10 μm. Spine density and total spine length are substantially reduced in 3-miR mix treated primary neurons compared to those treated with scramble RNA (n = 49-97 images) D. Hippocampal neurons were cultured in a multi electrode array (MEA) plate equipped with sixteen electrodes. Spontaneous activity of the neurons was recorded at every 3 hours (10 minutes/session) for 24 hours. Weighted mean firing rate, number of bursts, and network bursts are significantly decreased in neurons treated with 3-miR-mix compared to control. E. The aberrant neuronal firing activity (weighted mean firing rate) and reduced number of bursts and network bursts were observed across the 24 hour of time period.
Mbd2 is a direct target of miR‐290 cluster that represses glycolytic metabolism and reprogramming A ECAR screening of Dgcr8−/− ESCs introduced with siRNAs against selected targets.Figure 4A ECAR screening of Dgcr8−/− ESCs introduced with siRNAs against selected targets. B qRT-PCR and Western blotting analysis of Mbd2 expression in WT and Dgcr8−/− ESCs. C qRT-PCR and Western blotting analysis of Mbd2 expression in Dgcr8−/− ESCs transfected with miR‐294 mimic. D qRT-PCR and Western blotting analysis of Mbd2 expression in WT and miR‐290 cluster−/−ESCs. E Luciferase reporter assay for Mbd2 WT and Mut 3′ UTRs. Dgcr8 WT or miR‐294 reduced luciferase activity compared to Dgcr8−/− or CTR group, respectively. F RNA‐seq analysis for CTR‐ and miR‐294‐transfected Dgcr8−/− ESCs. Each point represents FPKM value for a given transcript. Only genes that were upregulated or downregulated greater than twofold in miR‐294‐transfected Dgcr8−/− ESCs were plotted. Genes that were also upregulated or downregulated greater than twofold in siMbd2‐transfected Dgcr8−/− ESCs are highlighted in red or blue, respectively. G Cellular glucose uptake, lactate production, and oxygen consumption rates were measured in Dgcr8−/− ESCs stably expressing Mbd2 shRNAs. H qRT-PCR and Western blotting showing that Pkm2 and Ldha were increased in Dgcr8−/− ESCs stably expressing Mbd2 shRNAs. I Cellular glucose uptake and lactate production were measured in Mbd2‐overexpressing Dgcr8−/− ESCs transfected with miR‐290 cluster. J Western blotting showing protein levels of PKM2 and LDHA in Mbd2‐overexpressing Dgcr8−/− ESCs transfected with miR‐290 cluster. A representative result is shown. K Glucose uptake and lactate production were measured in WT and miR‐290−/−ESCs transfected with Mbd2 siRNAs. L Western blotting showing protein levels of MBD2, PKM2, and LDHA in WT and miR‐290−/−ESCs transfected with Mbd2 siRNAs. A representative result is shown. M, N Oct4‐GFP MEFs were introduced with the indicated factors, and Oct4‐GFP‐positive colonies were counted on day 16 after virus infection. AP staining of iPSC colonies was shown below.
Figure 4PolyA-seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage. Cells were treated with siRNA against Fus (siFus) and/or U1 antisense morpholino (U1AS). Control cells (Cont) were treated with both control siRNA and control antisense morpholino. A. Correlation of changes in polyA site usage between U1-inhibition and Fus-silencing. Fold change in the RPM of a polyA site between siFus and Cont (siFus/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. B. Correlation of changes in polyA site usage between U1-inhibition alone and the combination of Fus-silencing and U1-inhibition. Fold change in the RPM of a polyA site between siFus+U1AS and Cont (siFus+U1AS/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. A slope of 1.0 indicates that Fus-silencing has no additive effect on U1-inhibition. Read distributions of RNAPII-FUS-tRIP (left) and RNAPII-U1C-tRIP (right) around APA sites repressed [the first quadrant in (A)] or activated [the third quadrant in (A)] by both FUS and U1 snRNP. The p-values for the differences between the repressed and the activated APA sites are indicated by circles. Arrowheads indicate noticeable peaks of RNAPII-FUS and RNAPII-U1C upstream to the repressed APA sites. Effects of Fus-silencing (pink line) and U1-inhibition (yellow line) on read distributions of RNAPII-FUS-tRIP (left) and RNAPII-U1C-tRIP (right) around APA sites repressed by both FUS and U1 snRNP [the first quadrant in (A)]. The p-values for the differences between contAS and U1AS (left panel), as well as those between siCont and siFus (right panel), are indicated by circles. Read distributions of U1A-FUS-tRIP (left) and U1C-FUS-tRIP (right) around APA sites repressed [the first quadrant in (A)] or activated [the third quadrant in (A)] by both FUS and U1 snRNP. The p-values for the differences between repressed and activated APA sites are indicated by circles. Arrowheads indicate noticeable peaks of U1A/U1C-FUS upstream to the repressed APA sites.
Figure 6A) Kaplan-Meier survival analysis (Log-Rank test with Holm-Sidak's correction for multiple comparisons). Mean age of death (± SEM) for G93A was 169.3 ± 2.5 days and for PKO/G93A was 185.6 ± 2.2 days, indicating that Parkin knockout prolongs lifespan of SOD1-G93A mice; n=19 for Non Tg (9 males and 10 females), n= 20 for PKO (10 males and 10 females), n= 18 for G93A (8 males and 10 females) and n= 19 for PKO/G93A (9 males and 10 females); p= 0.0000285. B) Body weight changes over time, relative to body weight at 45 days that is set at 100% for each mouse. Parkin knockout delays body weight loss in SOD1-G93A mice. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females); *p= 0.017 (at 150 days), *p= 0.004 (at 164 days), *p= 0.002 (at 171 days) and **p= 0.001 (at 178 days), by two-way ANOVA with repeated measures and with Holm-Sidak's correction for multiple comparisons. C) Clasping score changes over time. Parkin knockout does not affect clasping onset in SOD1-G93A mice. An intermediate score of 2.5 is indicated by the dotted line. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females). D) Representative images of NMJs in the soleus muscle at 130 days. Muscles were immunostained for neurofilament H (NF-H, in red) and stained with α-bungarotoxin (α-BTX, in green). Scale bar, 20 µm. E) Quantification of the percentage of innervated NMJs at 130 days indicates that Parkin knockout decreases denervation in SOD1-G93A mice. Results are expressed as mean ± SEM and were collected from n= 6 mice per group (3 males and 3 females); n of images evaluated = 49 for Non Tg (with 483 NMJs counted), 46 for PKO (396 NMJs counted), 47 for G93A (413 NMJs assessed) and 38 for PKO/G93A (325 NMJs counted); ***p= 0.0001, by unpaired Mann-Whitney test. F) Representative images of lumbar spinal cord sections at 130 days immunostained with ChAT. Scale bar, 200 µm. G) Quantification of ChAT-positive MN in the spinal cord ventral horn (dotted areas) at 130 days shows that Parkin knockout decreases MN loss in the spinal cord of SOD1-G93A mice. Results are expressed as mean ± SEM; n = 10 mice per group in Non Tg, PKO and G93A (5 males and 5 females) and n= 9 for PKO/G93A (4 males and 5 females); *p=0.031, by unpaired Student's t test. H H) Representative images of lumbar spinal cords sections immunostained for GFAP at 130 days. Scale bar, 200 µm. I) Quantification of the average GFAP staining intensity at 130 days indicates that Parkin knockout decreases spinal cord astrogliosis in SOD1-G93Amice. Results are expressed as mean ± SEM; n = 10 mice per group (5 males and 5 females); *p= 0.033, by paired Student's t test.
Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a) and transfected HeLa cells (b).f3Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a) Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b). VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d). e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux. f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux. g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h. h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.
Figure 4A - 5' end-labeled dgcM mRNA (10 nM) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram. When added, Hfq hexamers were at 50 nM, the sRNAs OmrA and OmrB at 250 nM. The accessibility of each nucleotide was probed with water (lanes 1-4, negative control for RNA integrity), RNase T1, or lead(II) acetate. Lane T1: RNase T1 under denaturing conditions. Lane OH: alkaline ladders. Binding sites of Hfq (green arrows and boxes), and site 1 (red arrows and boxes) and 2 (blue arrows and boxes) of the sRNAs, are highlighted. Note that site 2 is bound differently by OmrA and OmrB (blue boxes on gel). Black boxes indicate Hfq-dependent T1 cleavage enhancements. B - Secondary structure of dgcM mRNA ± Hfq, based on results in Figure 4A. Nucleotides are color-coded as in 4A. Unchanged T1 cleavages are indicated by filled arrows, enhanced T1 cleavages by arrow and star, protection from T1 cleavages by dashed arrows. Lead(II) acetate-protected sequences are indicated by full (Hfq+OmrA/B), dashed (Hfq + OmrA), or dotted (Hfq alone) frames. In the lower panel Q = Hfq, A = OmrA, and B = OmrB.
Figure 6A. Cdc20 binding assay in Xenopus egg extracts. The Cdc20-binding activities of WT APC/C and Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) were analysed as described in Fig 2A. B. Quantification of (A). The bar graph is quantification of bound Cdc20.The intensities of WT control at Ana-45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. C. Premature activation of a mutant APC/C (1-E562A) in Xenopus egg extracts. The purified recombinant WT APC/C or Apc-loop500-mutant APC/C (1-E562A) was incubated with its substrates (35S-labelled cyclin B and a version of cyclin B lacking the N-terminal 67 residues, ∆67) in APC/C-depleted (ΔAPC) interphase extracts supplemented with non-degradable cyclin B at 23˚C. Samples taken at indicated time points after addition of substrates were analysed by SDS-PAGE and autoradiography (Fig S7A). The relative cyclin levels are shown, normalized with reference to the intensities found at time 0 for each time point. Error bars, SEM from three independent experiments. A representative result is shown in Fig S7A. D. APC/C dephosphorylation after inactivation of CDK. The purified recombinant WT or Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) was incubated with APC/C depleted (ΔAPC) in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1h (Ana). After addition of p27 (0.3 µM), the samples taken at time points shown were analysed by SDS-PAGE and immunoblotting with indicated antibodies. 'Inter' denotes interphase.
Figure 3Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD) . Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls. Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S3A-D, one-way ANOVA followed by Tukey's multiple comparison test.
Dgcr8 loss leads to separable differentiation and survival defects in GPCsRT–qPCR of Dgcr8 exon 3 in Dgcr8Δ/Δ GPCs 5–7 days following tamoxifen treatment (n = 3).Figure 1RT-qPCR of Dgcr8 exon 3 in Dgcr8Δ/Δ GPCs 5-7 days following tamoxifen treatment (n = 3). Samples were normalized to actin, and data are presented relative to untreated Dgcr8flox/Δ GPCs. RT-qPCR for a panel of miRNAs in Dgcr8Δ/Δ cells 5-7 days post‐tamoxifen treatment (n = 3). Samples are normalized to U6 RNA. Representative images of GFAP immunofluorescence following 48 h of differentiation in control (R26 CreER + tamoxifen and untreated Dgcr8flox/Δ GPCs) and Dgcr8Δ/Δ GPCs. Representative Western blot of full length and cleaved caspase‐3 levels in control and Dgcr8Δ/Δ cells following 5−6 hours of differentiation (n = 2). Representative images of GFAP immunofluorescence following 48 h of differentiation in BakΔ/Δ;Baxf/f (control), BakΔ/Δ;BaxΔ/Δ (control), BakΔ/Δ;Baxflox/Δ;Dgcr8flox/Δ GPCs (control) and BakΔ/Δ;BaxΔ/Δ;Dgcr8Δ/Δ (TKO) cells (n = 3). RT‐qPCR for panel of astrocyte markers in control and BakΔ/Δ;BaxΔ/Δ;Dgcr8Δ/Δ cells (n = 3).
2019-nCoV-S employs TMPRSS2 for S protein priming.Fig. 3.priming.Ammonium chloride (A), were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins. E64d (CatB/L inhibitor) (B) and/or camostat (TMPRSS2 inhibitor) (B) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins. (C) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins. The average of three independent experiments is shown in panels A-C. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
Figure 5A An organoid culture genetically engineered using the CRISPR-Cas9 system to carry a BRAFV600E mutation represents a model for the serrated path to CRC. Upon TGFβ treatment, these culture gains a mesenchymal appearance (scale bars: 200 μm). B,C TGFβ stimulation does not induce (B) cleaved Caspase-3 or (C) BIM expression (n = 6) in the BRAFV600E-mutated organoid culture. B,C TGFβ stimulation does not induce (C) BIM expression (n = 6) in the BRAFV600E-mutated organoid culture. D,E Markers of the EMT program, (D) ZEB1 (n = 6) are strongly induced in the TGFβ-treated condition. D,E Markers of the EMT program, (E) FN1 are strongly induced in the TGFβ-treated condition. F,G FRMD6 is upregulated and CDX2 expression is reduced both on RNA (n = 6) as well as on protein level following TGFβ stimulation. F,G FRMD6 is upregulated and CDX2 expression is reduced both on RNA (n = 6) as well as on protein level following TGFβ stimulation. H TGFβ target genes downregulated upon TGFβ treatment of the BRAFV600E-mutant organoid culture (TGFβ down) are lower expressed in CMS4-SSAs compared to CMS1-SSAs and genes induced by TGFβ treatment (TGFβ up) are more strongly expressed in CMS4-SSAs than in CMS1-SSAs (n = 4 for CMS4-SSAs and n=8 for CMS1-SSAs; FC = fold change). I The differential expression of TGFβ target genes observed for CMS1-SSAs and CMS4-SSAs is maintained at the carcinoma stage for CMS4 versus CMS1 BRAF-mutant CRCs of the TCGA dataset (n = 4 for BRAF-mutant CMS4 and n = 34 for BRAF-mutant CMS1 CRC samples; FC = fold change).
Figure 4Sec61β wild-type (WT) and the 3R variant tagged at their N-terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon. Ribosome-nascent chain complexes (RNCs) were recovered using anti-FLAG affinity resin, beads were washed, bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS-PAGE and immunoblotted for endogenous rabbit SGTA, FLAG-tagged translation products, proteins of the 40S (Rps3) and 60S (Rpl17) ribosomal subunits or analysed by phosphorimaging (35S). The 3xFLAG peptide eluted material (lanes 4 and 5) corresponds to ~10% of the sedimented RNCs (lanes 7 and 8).