EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A and B) Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant. (C) Overview of 3-week-old plants. (D) Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants.)
Hwa interacts with ZNRF3.Figure 2A Schematic illustration of Flag pull-down assay. The empty vector (Flag-control) and Hwa-Flag were purified from HEK293T cells with anti-Flag beads and incubated with oocytes homogenate; associated proteins were identified by mass spectrometry. B Coomassie brilliant blue staining and MS analysis of Hwa-associated proteins after immunoprecipitation (Flag pull-down). Right (outlined text), thirteen matched peptide sequences that correspond to ZNRF3. Hwa-MF, Myc-Flag-tagged Hwa. C Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments. D Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-HA were immunoblotted with Flag and HA antibodies. Blots are representative of 3 independent experiments. E Colocalization of Hwa-Flag (red) and ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm. F Schematic diagram of ZNRF3 truncations. ZNRF3FL, full-length ZNRF3; ZNRF3ΔRING, ZNRF3 lacking the RING domain; ZNRF31-320, amino acids 1-320 of ZNRF3; ZNRF3320-606, amino acids 320-606 of ZNRF3; ZNRF3606-854, amino acids 606-854 of ZNRF3. G Co-IP of HEK293T cells transfected to express Hwa-Flag and vector only or plasmids encoding ZNRF3 truncation mutants as indicated in (F). Lysates immunoprecipitated with anti-HA were analyzed by immunoblotting with Flag and HA antibodies. Blots are representative of 3 independent experiments.
increases susceptibility to HFD- and lipid-induced insulin resistance.Figure 2Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Blood glucose levels and area under curve (AUC) during the glucose tolerance test. Blood glucose levels and AUC during the insulin tolerance test. Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Experimental procedure and hyperinsulinemic-euglycemic clamp (HEC) protocol. Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Percentage of suppression of hepatic glucose production (HGP). Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Experimental procedure for lipid infusion and the hyperinsulinemic-euglycemic clamp (HEC). Eight-week-old male WT and DOCK5-/- mice Glucose infusion rates (GIR) during lipid infusion.
Figure 4A, B γH2AX/TelG immuno-FISH (A) revealed increased TIF formation in IMR90 cells transduced with DAXX-targeted lentiviral sgRNAs (sgD#1 and #2) (B). Scale bar, 10 µm. Arrows denote telomeres that are positive for γH2AX signals. (B) Percentage of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test. C Western blots show depleted DAXX protein expression in Ad-Flp-infected cd-DAXX cells. D, E PML/TelG immune-FISH (D) show enhanced APB formation in Ad-Flp infected cd-DAXX cells (cd-D#1 and cd-D#2) (E). The cells were analyzed at day 18 post Ad-Flp infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (E) Percentages of APB+ cells are expressed as means ± s.d., N = 3, unpaired t test. Depletion of DAXX induced delayed onset of growth arrest (F) (F) 2,000 cells were plated at day 4 post Ad-Flp infection. Data are expressed as means ± s.d., N = 3. Depletion of DAXX induced C-circle formation (G).
Figure 4FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 embryoids specified in the presence of 4mM dm-αKG and PGC cytokines. Representative flow cytometer profiles are depicted. Graphs show the average fractions of GFP+ cells from duplicate experiments. P1-GFP, Prdm1-GFP. FACS analysis of Prdm1-GFP positive (GFP+) cells in day-2 embryoids aggregated under addition of LIF (10ng ml-1) and BMP4 (500ng ml-1), dm-αKG (4mM), or DMSO. Representative flow cytometer profiles are displayed. Average fractions of GFP+ cells are calculated from duplicate assays. P1-GFP, Prdm1-GFP. FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 PGCLC aggregates specified from 4mM dm-αKG-treated (t=48h to t=72h) EpiLCs. Representative flow cytometer profiles are depicted. The average fractions of GFP+ cells, quantified from triplicate experiments, are shown. P1-GFP, Prdm1-GFP. Transcript analysis by qRT-PCR of PGC specifiers, demethylating enzymes, mesoderm, endoderm, and ESC regulators in FACS-sorted day-4 Prdm1-GFP embryoids induced from 4mM dm-αKG-treated 72h EpiLCs. Expression levels are normalized to Prdm1-GFP negative cells from control embryoids. Graphs represent averages from triplicate experiments. +, Prdm1-GFP positive cells; -, Prdm1-GFP negative cells. Western blot analysis for H3K9me2, H3K27me3, and DNMT3b in 4mM dm-αKG-treated EpiLCs. H3 is used as a loading control. 48h, dm-αKG and DMSO, respectively, supplementation from t=24h to t=48h during the EpiLC differentiation. 72h, dm-αKG and DMSO, respectively, supplementation from t=48h to t=72h during the EpiLC differentiation. ChIP-qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state (Esrrb, Arid5b) and PGC fate (Tfap2c), respectively, in naïve ESCs and at t=48h following EpiLC induction in the presence of 4mM dm-αKG and DMSO, respectively. Graphs show enrichment of H3K9me2, H3K27me3, and IgG control, respectively, relative to DMSO-treated EpiLCs. Model illustrating the extension of the transient developmental competence for the PGC fate through αKG.
Fig.Figure 4(A) Time-lapse images of PCNA-mRuby tagged A2dd cells. The imaging sequence was started at the time of Doxycycline addition, and degron activity was triggered three hours later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min, scale bar equals 10µm. For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase. (B) Single-cell analysis of 40 cells per condition treated each line represents the timing of G2/M/G1 progression for a single cell. (C) A2dd cells were pulsed with EdU to label S-phase then protein degradation was induced by DIA addition for 5h and before fixation and Edu/CENPF/DAPI staining. Cells that were in G2 at the time of DIA or mock treatment were identified as CENPF positive, EdU negative. Mitotic cells were excluded based on nuclei morphology. The bar plots show mean percentage of EdU negative/CENPF positive cells of three repeats, error bars indicate standard deviation. (D) Images from time-lapse sequence of mitosis showing cell division in controls and after addition of 0.5µM PD-166285 in DIA treated A2dd labelled with SiR-DNA (time is indicated as hh:min, the scale bars represent 20µm). (E) Accumulative index of mitotic entry measured by time-lapse microscopy in A2dd cells following mock or DIA treatment and addition of Wee1 inhibitors MK1775 (1µM) or PD-166285 (0.5µM). Asynchronous cells were DIA treated for four hours and then imaged for six hours in five-minute intervals. (F) Quantification of metaphase duration in A2dd cells treated with DIA and Wee1 inhibitors as indicated (n>50 per repeat, the boxplot indicates median, first and third quartile and minimum/maximum values. (G) A2dd cells were transfected with Ctr or B55α/δ siRNA. 48 hours after transfection the cells were treated with Thymidine for 24 hours and then released. 10 hours later the cells were probed for B55 depletion by immunoblotting using a pan-B55 monoclonal antibody. (H) Lack of B55 partially rescues mitotic entry in DIA treated A2dd cells. Cells transfected with Ctr siRNA or B55α/δ siRNA were released from Thymidine, mock or DIA treated and analysed by widefield live-cell imaging eight hours after the release. The cells were imaged every 10 minutes for 15 hours and manually scored for mitotic index.
Figure 5A. Fluorescence micrographs of two different COPI•COPII BiFC pairs are shown: Sec16pVN•α-COPVC or Sec16pVN•ε-COPVC. DSL1 cells show dispersed punctate patterns resembling ER exit site patterns. In the dsl1-5xWA mutant, cells exhibit additional bright spots. Scale bar 10 µm.B. Quantification of fluorescence intensity differences of the strains shown in (A). COPI•COPII BiFC fluorescence is significantly increased in cells carrying the dsl1-5xWA mutant as compared to DSL1 wild type. Mean values + SEM of at least nine independent experiments (n=918) are displayed (two-sample unpaired twotailed ttest). C. Time-lapse micrographs of cells expressing the β'-COPVN•Dsl3pVC BiFC pair in a dsl1-5xWA background. Cells were imaged embedded in agarose at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Fluorescence signals retain the characteristic polarization pattern as seen DSL1 wild type cells (see Fig 3). Scale bar 10 μm.
Figure 3A. HEK293 cells treated for 4 h with 100 μM H2O2. Phospho-histone H2A.X (γH2A.X) was assessed by immunofluorescence analysis. Nuclei (blue) were stained with Hoechst 33342. scale bar: 10μM. 4X digital magnification is shown at the bottom left of each image. B. Additionally, γH2A.X was also assessed by Western blot at the indicated time points. GAPDH was used as loading control (top panel). Densitometry of each lane intensity is normalized to GAPDH, selected as loading control, and expressed as % of γH2A.X in H2O2-treated versus untreated cells. Values represent the means ± SD of two independent experiments (bottom panel). C. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of ATR/CHK1 axis activation. Basal and phospho-active forms of ATR and CHK1 (at Ser345) were assessed by Western blot. GAPDH was used as loading control. D. HEK293 cells were treated for 8 h with 20 μM neocarzinostatinE. HEK293 cells were treated for 2, 4 and 8 h with 20 μM neocarzinostatin (NCS). After treatment, cells were incubated with 2',7'-H2DCF-DA to cytofluorometrically assess the intracellular production of H2O2. Values are shown as units of DCF fluorescence relative to untreated cells. Values are shown as units of fluorescence relative to NCS-untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. n.s., not significant. F. HEK293 cells were transfected with plasmids coding for the wild-type (WT), C2991L redox mutant (CL) of ATM, or with an empty vector (Empty) (used as negative control) for 40 h and treated for additional 8 h with 100 μM H2O2. Basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot analysis. Vinculin and GAPDH were used as loading controls. Two different exposures (low and high) were selected to highlight differences in P-ATM levels in different experimental settings.
FYCO1 can dimerize and is recruited to intracellular membranes in an FYVE domain–dependent manner.Figure 5.(E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection. (B) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles. HeLa cells were imaged 24 h after transfection with the indicated constructs. (C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation. (D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein-lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVEFYCO1, MBP-2xFYVEFYCO1, or MBP-FYCO1863-1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (E) The construct containing a tandem fusion of two FYVE domains from FYCO1 (2xFYVEFYCO1) localizes to the nuclear envelope and ER in HeLa cells. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A and E) Insets show an enlarged field of interest. (F) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs (left) and PXp40phox (right). HeLa cells were imaged 24 h after transfection with the indicated constructs. Bars, 10 µm.
Figure 6Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm. (Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant. Model of NuRD function during differentiation of pluripotent cells. NuRD facilitates lineage commitment of ES cells after exit from pluripotency (red arrows), allowing cells to form differentiated cell types (Top). In the absence of Mbd3, residual NuRD activity ensures cells retain the appropriate differentiation trajectory, but the cells are unable to reach a differentiated cell fate (dotted red arrows; Middle). In the absence of all three MTA proteins there is no residual NuRD activity and ES cells are unable to either achieve appropriate lineage commitment, or to maintain the proper differentiation trajectory (dotted arrows, Bottom).
CRMP4-dynein interaction is enhanced in ALS motor neuron axons (A) Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in SOD1G93A compared to WT P90 sciatic nerves under physiological conditions.Figure 5(A) Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in SOD1G93A compared to WT P90 sciatic nerves under physiological conditions. IgG antibody was used as a control. (B) Quantification of the blot in A. 3 repeats, 12 sciatic nerves per condition were used in each repeat. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Data presented as mean ±SE (Ratio Paired t-test, *p = 0.0416). (C) Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in Cos7 cells that were transfected with mutant CRMP4 I141V compared to control. IgG antibody was used as a control. (D) Quantification of four repeated pull down in C. The CRMP4 intensity band was normalized to the DIC intensity band for each repeat. (Student's t-test, n=4, Data presented as mean ±SE, *p=0.0393). (E) Representative images from the proximity ligation assay (For explanation of PLA technique; please refer to method section) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment. Scale bar: 5µm. (F) Quantification of the CRMP4-DIC puncta number per primary motor neuron axon in each condition. We analyzed ~20 axons per condition from 3 independent chambers per group (One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.01 *p=0.04) (G) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human-derived proximal axons post peptides treatment, Sema3A treatment, Sema3A + peptides treatment or untreated controls. Scale bar: 5µm. (H) Quantification of the CRMP4-DIC puncta number per axon in healthy or C9orf72 human-derived MN proximal axons after Sema3A treatment, Sema3A + peptides treatment or untreated controls. Data collected from 3 independent chambers in each condition. Total of 37 healthy untreated proximal axons, 61 healthy proximal axons with peptides treatment, 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment. 67 C9orf72 untreated proximal axons, 63 C9orf72 proximal axons with peptides treatment, 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored. Data presented as mean ±SE. One-way ANOVA, Tukey's multiple comparisons test, n=3, ****p<0.0001.
Figure 2A RT-qPCR analysis of lincRNA-EPS transcripts in the lincRNA-EPS-stably-overexpressed iBMMs (lincRNA-EPS) and corresponding control cells (EV). B LincRNA-EPS iBMMs were infected with VSV (MOI 1) for 8 h, VSV titers were measured by plaque assay. C, D The EV and lincRNA-EPS iBMMs were infected with SeV (MOI 1) (C) or HSV-1 (MOI 5) (D) for 12 h. The viral RNA was measured by RT-qPCR. E The transcripts level of lincRNA-EPS in control (sgCtrl) and lincRNA-EPS knockdown (sglincRNA-EPS) RAW264.7 cells were measured by RT-qPCR. sgCtrl and sglincRNA-EPS RAW264.7 cells were infected with VSV-GFP (MOI 0.1) for 6 h or infected with VSV (MOI 0.1) for 8 h. The fluorescence of GFP was checked by microscope (F) G sgCtrl and sglincRNA-EPS RAW264.7 cells were infected with VSV-GFP (MOI 0.1) for 6 h or infected with VSV (MOI 0.1) for 8 h. the viral titer of VSV from the cell supernatant was measured by plaque assay (G). H, I sgCtrl and sglincRNA-EPS RAW264.7 cells were infected with SeV (MOI 1) for 8 h (H) and HSV-1 (MOI 5) for 12 h (I). The viral RNA was measured by RT-qPCR.
Figure 3(A) Schematic representation of the reporter constructs used for analysis of protein biogenesis by flow cytometry. All constructs contain an N-terminal GFP and a C-terminal RFP, separated by a viral 2A peptide that mediates peptide bond skipping. Changes in the stability of SQS, Xport-A and Xport-A4L fused to RFP change the RFP:GFP fluorescence ratio. (B-D) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC5KO+EV" indicates a knockout of EMC5 stably harbouring empty vector, while ''EMC5KO+EMC5" indicates EMC5KO cells rescued by inducible re-expression of a stably integrated EMC5. (E-G) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC6KO+EV" indicates a knockout of EMC6 stably harbouring empty vector, while ''EMC6KO+EMC6" indicates EMC6KO cells rescued by re-expression of a stably integrated EMC6.
Figure 5A Real-time RT-PCR analysis of mRNA levels of ER-GATA-1 target genes and two housekeeping genes, Eif2s1 and Eif3k (n = 4, mean +/- SE).B Fold changes of mRNA levels after 5-ALA treatment relative to no treatment in β-estradiol-treated WT clone 1 or double-mutant clone 1 (n = 4, mean +/- SE). C Real-time RT-PCR analysis of primary transcripts of ER-GATA-1 target genes (n = 4, mean +/- SE).D Fold changes of primary transcripts with 5-ALA treatment in comparison with no treatment in b-estradiol-treated cells (n = 4, mean +/- SE). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
βOHB promotes PDA aggressivenessA Tumor weight in KIC mice treated with βOHB (100mg/kg/day, i.Figure 1A Tumor weight in KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=13 mice for NaCl and n=15 mice for βOHB). Data are expressed as mean ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05. B Kinetic of spheroid area from PK4a cells cultured during 12 days in medium alone (untreated) or with βOHB 1 or 10mM (n=4, and 5 respectively). Data are expressed as mean ± SEM. Significance compared to untreated cells was defined by two-way ANOVA followed by a Dunnett's multiple comparisons test. **p<0.01, **** p<0.0001. C Representative images of mouse PDA and human PDA primary cells-derived organoids after 7 days of culture in medium alone (untreated) or supplemented with 1 or 10mM of βOHB. Scale bar: 1000 µm. D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm. F Histological characterization of liver and spleen of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=14 mice/group). Number of mice displaying metastatic liver or spleen in each experimental group is reported. G Schematic showing isotopomer transition from [U-13C]βOHB to label TCA-cycle intermediates, glutamate and proline. Grey filled circles indicate 13C carbon derived from labeled βOHB. Empty circles illustrate unlabeled 12C-species. H [U-13C]βOHB tracing into the TCA intermediate: citrate in poorly differentiated PDA explants from KIC mice (n=5). Data are expressed as mean ± SEM. I Schematic showing ketone metabolism pathway (left panel). Reversible enzymes involved in the production of acetyl-CoA, acetoacetyl-CoA, acetoacetate, βOHB, mT, SCOT1/2, and BDH1/2 are indicated in bold case. Immunoblots of ketone metabolic enzymes (BDH1/2, SCOT1/2, mT) (right panel) in pancreatic tissues from 7-week old KI (n=3 for BDH1/2, SCOT1/2, and n=4 for mT) and KIC mice (n=4).
Figure 2(A-C) Three different mediation models are shown, testing plasma P-tau217 as a statistical mediator of plaque density on tangle density. Panel A shows the mediation of total tangle density (significant mediation, 60% of the direct effect of plaque density on tangle density was explained by plasma P-tau217). Panel B shows the mediation of medial temporal lobe (MTL, entorhinal cortex and hippocampus) tangle density (no mediation). Panel C shows the mediation of tangle density in all regions except the MTL (strongest mediation, 77%). The P-values are from linear regression models used to estimate the mediation effects.
AICAR induces AMPK phosphorylation of raptor and the recruitment of 14-3-3 to the ULK1-mTORC1 complex.Figure 4  293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies.