EMBO/sd-character-level-ner huggingface dataset:
Figure 8A. Electropherograms of the sequences containing the mutations observed in family E952. Position within the OTOF cDNA (NM_001287489.1) is indicated above each sequence. For each mutation, the wild-type sequence and the heterozygous sequence are shown. For purposes of clarity, the two alleles carried by subject E952-II:1 were subcloned into a plasmid vector and sequenced in separation. Sequences of the subcloned mutant alleles are shown in the bottom row. B. Pedigree of family E952 showing the segregation of mutations in the OTOF gene. C. Apparent decrease in insertion efficiency for the TMD mutant version. While the WT otoferlin glycosylation amounted to 3.84 ± 0.53, the TMD mutant had only 2.13 ± 0.65 (n=3, p<0.05). Data are represented as mean ± SEM.
The activity of ATP synthase determines lateral, radial and transmembrane pH gradients.Figure 8A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions. B) Mitochondrial pH at CV SU γ measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions. C) pH in the matrix bulk (MPP60 as mitochondrial targeting sequence) measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions. D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions. E) pH differences between SU e and SU γ of ATP synthase SU e is part of FO, SU g of F1. Triple stars in columns indicate that the difference between the compared pH values constituting the ∆pH was significant; p≤0.001. F) Radial pH gradient between matrix bulk and subunit γ of ATP synthase. G) pH difference between SU e of ATP synthase and matrix bulk. H) Transmembrane pH difference between local pH at CIV (CoxVIIIa) and matrix pH. I) Lateral pH gradient between CIV SU CoxVIIIa and CV SU e. Be aware that the pH under glycolytic conditions at SU e was lower than the pH at CIV in the ICS, resulting in a "negative" ∆pH. (A-I) One-way ANOVA comparison of sets of pH values at the indicated localizations. Box (75%) and whisker plots, median (horizontal line in box), mean (box in box). p≤0.001: ***, p≤0.01: **, p≤0.05: *; n.s.: non-significant (N=3). Statistics: one-way ANOVA with post hoc Schéffe test for normal distribution. J) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ. The pH was color coded according to respective emission ratio. The pH at SU γ in IF1-H49K-expressing cells is compared with WT cells. (Scale bar: 1 µm). K) Schematic drawing showing the resolved pH values and resulting gradients under respiratory and glycolytic conditions. The color gradient was generated following the ratiometric pH code used throughout the study.
Figure 5(A) Putative miR-2137 binding site on 3` UTR of human (Gene ID 3636), chimpanzee (Gene ID 451403), rat (Gene ID 65038) and mouse (Gene ID 16332) INPPL1 mRNA. (B) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates). (C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates). (D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).
Calyculin A treatment reverses the effect of LUZP1 on di-phosphorylated myosin light chain (ppMLC) levels.Figure 4Representative confocal micrographs of co-cultures of wild-type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells [40.87 ± 9.95 arbitrary units (a.u.) (WT) vs. 39.48 ± 6.04 a.u. (LUZP1 KO)]. n = 3. p = 0.54 (Mann-Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio [1.00 (1st lane, control) vs. 1.13 ± 0.24 (2nd lane) vs. 1.01 ± 0.44 (3rd lane) vs. 1.08 ± 0.73 (4th lane)]. n = 4. p = 0.49 (Kruskal-Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co-cultures of Venus-LUZP1-expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells [control, 21.14 ± 16.80 a.u. (WT) vs. 3.10 ± 1.72 a.u. (LUZP1 KO); calyculin A, 25.24 ± 10.54 a.u. (WT) vs. 20.65 ± 5.62 a.u. (LUZP1 KO); washout, 22.09 ± 7.90 a.u. (WT) vs. 7.92 ± 4.01 a.u. (LUZP1 KO)]. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.
Figure 3Schematic representation of the experimental procedure to obtain the proteomes of U2OS control cells (-Dox) or cells inducible expressing FLAG-HA-FAM134 proteins upon 24hrs doxycycline induction (+Dox) by mass spectrometry (MS). Cells were grown in basal condition or starved with EBSS for 8hrs prior to lysis. Principal Component Analysis of the MS data after indicated treatments. Hierarchical clustering of label-free quantitation (LFQ) intensities of significantly changed proteins (ANOVA, FDR 0.01). Cluster framed in green includes proteins with a "endoplasmic reticulum" enriched GO. Profile-plot of protein levels (LFQ intensities) of the cluster enriched for "endoplasmic reticulum" upon indicated treatments. List and frequency of GOCC terms related to the protein cluster in (D). CANX, REEP5 and SEC22B as sample proteins extracted from (D). Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8. ER-phagy flux, represented by the RFP/GFP ratio (total fluorescent intensities), upon basal (DMSO) or autophagy induced (Torin1) conditions, in U2OS control cells and cells overexpressing FAM134 proteins upon 24hrs Dox induction. Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3 Comparison of DMSO or Thapsigargin (Tg) treated U2OS ssRFP-GFP-KDEL cell lines overexpressing indicated FAM134 family members upon 24hrs Dox induction.
UBTD1 regulates YAP ubiquitylation by modulating its association with β-TrCP (A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1.Figure 4(A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. YAP was used as bait. The IgG isotype was used as a negative control. (B) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. UBTD1 was used as bait. The IgG isotype was used as a negative control. (C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. (D) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c and UBTD1. UBTD1 was used as bait. IgG isotype was used as a negative control. (E) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c, YAP and β-catenin. DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single). β-TrCP was used as bait. IgG isotype was used as a negative control. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Proximal ligation assay monitoring (F) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image. quantification (G) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). (H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion.
Accelerated disease onset in Sig1R-deficient SOD1G85R miceA.Figure 3A. The levels of Sig1R in the lumbar spinal cord (LSC) or brain of Sig1R−/−, Sig1R+/−, or Sig1R+/+ mice at 5 months old. Representative immunoblots obtained from three independent experiments are shown. Asterisk denotes non-specific bands. B and C. Sig1R-deficient SOD1G85Rmice (SOD1G85R/Sig1R−/−) exhibited accelerated onset of the disease (B) and shortened survival time (C) compared to SOD1G85Rmice with one or two copies of Sig1R (SOD1G85R/Sig1R+/−, SOD1G85R/Sig1R+/+). Open circles indicate Sig1R knockout mice (Sig1R−/−) that never developed motor neuron disease but that were sacrificed at 396 days of age (n = 8). Mean onset or survival times of mice with ±SD are shown on the top of Kaplan-Meyer curves. **: p < 0.01, *: p < 0.05, log-rank test. n = 14 (SOD1G85R/Sig1R+/+), 29 (SOD1G85R/Sig1R+/−), 17 (SOD1G85R/Sig1R−/−). D and E. Body weights were lost earlier both in male (D) and female (E) of SOD1G85R/ Sig1R−/−mice. Data are shown as mean ± SD. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. n = 8 (SOD1G85R/Sig1R+/+), 13 (SOD1G85R/Sig1R+/−), 8 (SOD1G85R/Sig1R−/−) in male (D), and n = 6 (SOD1G85R/Sig1R+/+), 16 (SOD1G85R/Sig1R+/−), 9 (SOD1G85R/Sig1R−/−) in female (E). The used animals were the same in the panels B and C. The animals used in the panels D and E were the same in the panels B and C. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. Two-way ANOVA with subsequent post hoc Tukey's test. F. Decreased performance on the rotarod test was observed in SOD1G85R/ Sig1R−/−mice. The test was performed at 0-30 rpm with 0.1 rpm/s acceleration every week. Data are shown as mean ± SD. **: p < 0.0001 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, † †: p < 0.0001 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. The used animals were the same in the panels B-E. Two-way ANOVA with subsequent post hoc Tukey's test. G and H. Motor function of WT (Sig1R+/+) and Sig1R knockout (Sig1R−/−) mice at 5 or 12 months old was evaluated by the rotarod test (0-30 rpm, accelerated at 0.1 rpm/s). Average time on the rotating rod was plotted. Error bars denote SD. Unpaired t-tests.
Figure 3Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM. Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM. (C) Quantification of Piezo2 current inactivation rate (Tau) in HEK cells transfected with Piezo2+D2RS-YFP. Data are shown as mean ± SEM and scatter plots (n=13) (D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM. (F) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=5, Paired t-test). Data are shown as mean ± SEM and scatter plots. (G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM. (I,J) Quantification of MA current inactivation rate (Tau) measured for large DRG neurons dialyzed with Gβγ (n=7, **P<0.01, Paired t-test) and vehicle (Paired t-test). Data are shown as mean ± SEM and scatter plots.
Figure 1Western blotting showing the expression of RAP1 and TRF2 in MRC-5 cells of different population doublings (PD). Senescent cells (PD 72) were left in culture for at least 4 weeks before harvesting for analysis. ChIP analysis of young (PD 30) and senescent (PD 72) MRC-5 cells performed with either anti-RAP1, anti-TRF2 or an IgG antibody. The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe, stripped and hybridized to an Alu probe in order to determine unspecific binding. Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input. Data represent mean +/- SD of three biological replicates. Immunofluorescence detection of 53BP1 (red) and a FISH probe staining telomeres (green) in young (PD 26), pre-senescent (PD 66) and senescent (PD 72 + 4 weeks) MRC-5 fibroblasts. The upper graph shows the percentage of telomere co-localizing with 53BP1 (TIFs). Total DNA damage was measured by immunofluourescence detection of 53BP1. The lower graph shows the total number of 53BP1 foci per nucleus. Approximately 40-50 cells were analyzed per replicate and per condition. Bars represent SEM of 2 biological replicates.
Figure 11Under normal growth conditions, MPK3/6 are less active and ARR1/10/12 maintains optimal cytokinin response to control plant growth and development. Exposing plants to high salinity activates MPK3/6, which interact with and phosphorylate ARR1/10/12. Phosphorylated ARR1/10/12 are degraded through the 26S proteasome-dependent pathway, eventually repressing the cytokinin response and leading to growth inhibition. X represents inhibited signaling or process.
Figure 1Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (plasma: n=64; saliva: n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgG to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=12, LoD (RBD)=13.8. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgG concentration of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU. The box plots show the interquartile range, the horizontal lines show the median values, and the whiskers indicate the minimum-to-maximum range.
| Kugeln do not interact with brain lymphatic endothelial cells (BLECs) in Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916, nor do they take up injected IgG-conjugated Alexa 674.Figure 5Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916. BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads). No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats). Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads). Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead). Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test). Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test). Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).
Figure 2A. ATP quantification on aging from 3 days to 1 year in Ifnb+/+ and Ifnb-/- midbrains. Error bars are mean + SEM from 6 mice. B. ATP amounts in DIV18 CNs, untreated or complemented with 30 U/mL rIFN-β. Error bars equal SEM from 5 (untreated cells) or 3 (treated cells) independent experiments. C. ATP quantification on total mitochondrial mass in DIV6 CNs. Error bars show mean + SEM from 6 independent experiments. D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE. F. Quantification of the respiratory control ratio (RCR), calculated as the ratio between S3 and S4o. G. ROS levels, based on mean fluorescence intensity of DCFDA in DIV18 Ifnb+/+ and Ifnb-/- CNs, cultured with or without 30 U/mL rIFN-β. Error bars equal SEM from 3 independent experiments. H. DCFDA quantifications in δIfnb N2A cells on treatment with the mitochondria-specific antioxidant S3qet and Mitotempo. I. MitoSox quantification in DIV6 Ifnb+/+ and Ifnb-/- CNs. Rotenone was used as a positive control for the assay. J. Expression of oxidative stress response genes by qPCR in DIV6 Ifnb+/+ and Ifnb-/- CNs. K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 microns. L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5-3 months) and old (aged 6-12 months) Ifnb+/+ and Ifnb-/- mice. M. Immunoblot for optineurin in 6-week-old midbrains of Ifnb+/+ and Ifnb-/- mice. N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without 30 U/mL rIFN-β.
Figure 4A. Co-immunoprecipitation of ectopic IN1 using TAP-tagged-A190 as bait from yeast protein extracts expressing WT or the indicated IN1-HBH mutants from a pTet-Off promoter in the absence of doxycycline. Expected sizes are 204 kDa for A190-HA and 82 kDa for IN1-Strep (WT and mutants). B. Genome browser visualization of HA-IN1 occupancy at the RDN1 locus. Occupancy of WT HA-IN1 and K617A, S621A and L622A HA-IN1 mutants is represented in each panel. Control, as described for panel 3D. C. Top. IN1 is recruited at Pol I-transcribed genes. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the RDN1 locus with DNA amplicon positions. D. Recruitment of WT and mutant HA-IN1 (K617A, S621A, or L622A) at the 18S rDNA locus transcribed by Pol I. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3).
AMPK and mTORC1 interact with ULK1 through the PS domain and the kinase domain of ULK1, respectively.Figure 2(A) A schematic of ULK1 illustrating its kinase domain, proline/serine-rich domain, C-terminal (CT) domain, and regions for binding to AMPK and mTORC1. (B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.
Figure 2Localization of Ams1 in WT, lap2Δ, ape2Δ, and ape4Δ cells. Scale bar, 3 μm.   Localization of Ape4 in WT, lap2Δ, ape2Δ, and ams1Δ cells. Scale bar, 3 μm.   Ape2 but not Ams1 or Ape4 co-immunoprecipitated with Lap2. Endogenously TAP-tagged Lap2 was immunoprecipitated. Ams1 was endogenously tagged with a 13Myc tag. Nbr1 but not Lap2 or Ape2 co-immunoprecipitated with Ams1. Endogenously mCherry-tagged Ams1 was immunoprecipitated. Nbr1 was endogenously tagged with a TAP tag. Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.
Figure 3(A) Schematic representation of HsSpindly. The position of a CC1 box (red) and of the Spindly box (green) required for binding Dynein:Dynactin are shown, together with the position of relevant predicted coiled-coil regions. (B) Cartoon representation of the 7-bladed ROD β-propeller (indicated as blades 1-7) modelled with AF2. The positions of blade 2, blade 3, and an α-helical hairpin representing an insertion between strands β3c and β3d are indicated. The four strands of each blade are indicated as A-D, with A and D being the innermost and outermost strands. The circle represents the entry point of the farnesyl binding pocket. Surface representation of the ROD β-propeller. The circle is in the same position shown in panel A. The position of Leu120 is shown in yellow. This residue was targeted by photoactivatable crosslinker groups introduced in the farnesyl group attached to Cys602 of Spindly (D) The position of a modelled peptide corresponding to the C-terminal region of Spindly as predicted by AlphaFold2 with a farnesyl moiety modelled on Cys602. (E-F) A farnesyl moiety, shown in cyan spheres together with a few C-terminal residues of a modelled Spindly peptide, fitted snugly into a pocket lined exclusively by the side chains of several hydrophobic residues. (G) Size-exclusion chromatography profiles and corresponding SDS-PAGE of the indicated samples. Two mutant RZZ complexes containing mutations in ROD L110F-L119F-L120K or I191M are indicated respectively as FFK and I/M. Note that in the bottom SDS-PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker. For all other shown SDS-PAGE gels, the marker precedes the first fraction.