EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) 4D9 demonstrates standard IgG pharmacokinetics in vivo. Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours, 4 and 7 days after 10 mg/kg intravenous injection of antibody. (B) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg/kg and 4D9-hIgG dosed intravenously at 100, 50 and 10mg/kg. Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM (n=5). (C) Schematic diagram depicting the target engagement assay setup for bound and total sTREM2 detection. For the bound assay, a secondary anti-human IgG detects 4D9 antibody bound to soluble TREM2 in CSF and plasma. For the total assay, a saturating amount of 4D9 antibody is added to eliminate unbound sTREM2. The ratio of 4D9-bound sTREM2 to total sTREM2 is calculated to determine the level of target engagement achieved by the concentration of antibody present. (D) Target engagement timecourse demonstrated near 100% 4D9-bound sTREM2 in CSF of wildtype mice at 24 hrs post dose. Over a 10 day timecourse with timepoints at day 1, 2, 4, 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ~50% by day 10. Animals were dosed intravenously with 50mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM (n=5). (E) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg/kg with >50% bound sTREM2 at 1mg/kg of antibody. 4D9-bound sTREM2 was undetectable at 0.1mg/kg and lower. 4D9 was IV-dosed at 50, 10, 1, 0.1, 0.01, 0.001, 0.0001 mg/kg, and isotype dosed at 50mg/kg. CSF bound: total sTREM2 in wildtype mice was measured at 24hrs. Data represent the mean ± SEM (n=5). (F) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD-platform based ELISA. Data represent the mean ± SEM (n=5) and are shown as pg TREM2 per ug of total protein. One-way ANOVA, Dunnett's post hoc test, p (isotype vs 4D9 (100mg/kg)) < 0.0001; p (isotype vs 4D9 (50mg/kg)) = 0.0007.
Figure 1(A) BDR2637 (Pveg-mCherry) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry signal (purple) and phase contrast (grey) of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm. Corresponds to Movie EV1. (B) Plaque formation was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected PY79 (WT) (10-6 PFU/ml) cells spread over an MB agar plate. Shown are images captured at the indicated time points (hrs). Scale bar 2 mm. Corresponds to Movie EV2. (C-D) The dynamic of SPP1 (C) and Phi29 (D) plaque formation following PY79 (WT) infection was monitored as described in (B). Shown is the diameter of individual plaques for each phage (n ≥ 12), with the average highlighted in red.
Figure 5A Hair cell viability in larval zebrafish (assessed by DASPEI staining) following cisplatin treatment at the indicated concentrations. Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons between cisplatin concentrations (A) were assessed by one-way ANOVA. ; **, P<.01; ****, P<.0001 (Tukey test). B Hair cell viability in larval zebrafish pre-treated with control-, tlr4ba- and/or tlr4bb-targeting morpholino oligonucleotides (MO) and subsequently treated with 15 μM cisplatin. Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons to control morpholino (B, except as indicated in blue) were assessed by one-way ANOVA. *, P<.05; **, P<.01; ****, P<.0001 (Tukey test).
Figure 3A. Effects of selected kinase inhibitors with differential effects on Rb∆K7 vs. control MEFs from initial kinome screens. Average proliferation rate of untreated MEFs is shown on the right (white column). Arrows point to pan-CDK- and CDK2/7/9-inhibitors. Bars represent mean ±SD, n=3 independent MEFs. B. Increased resistance of Rb∆K7 vs. control MEFs to CDK2 inhibition (P=0.008 by two-tailed unpaired student's t-test). Effect of a pan CDK inhibitor was near significance (P=0.07). Bars represent mean ±SD, n=3 independent MEFs. C. Left, representative flow cytometric analysis with 7-AAD and AnnexinV, showing a significant decrease in apoptosis in Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs. D. Increased G1 phase in Rb∆K7 MEFs. Mean ±SD of propidium Iodide flow cytometry analysis showing a significant increase in G1 and decrease in S phase in synchronized Rb∆K7 MEFs relative to control (n=3 independent MEFs). E. Representative bright field images of multi-passage MEFs showing immortalized control cells but large senescing Rb∆K7 cells (arrows, original magnification: 200x; n=4-5). F. Representative images showing increased senescence in Rb∆K7 MEFs (arrows) determined by senescence-associated β-galactosidase assay (SA-βGAL; original magnification: 200x; P=0.0395 by two-tailed unpaired student's t-test, n=4 technical replicates
a) The gene structure of Rae1.figf2(a) The gene structure of Rae1. The black-arrowed boxes indicate exons. A transposable element, P{GawB}NP3499, located ∼400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1. PCR analysis indicates that ∼900 bp and a ∼1.5-k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12, respectively. (b) Western blot analysis on Rae1 protein in the brain lysate of wild-type, Df(2R) 5764/+, Rae1EX28/+, or late second instar Rae1EX28, Rae1EX28/Df(2R) 5764(/Df) and Rae1EXB12 /Df(2R) 5764 larvae. β-tubulin was used as the loading control. Full-length blots are presented in Supplementary Figure 9. (c) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains. Rae1 protein levels were first normalized to β-tubulin protein levels and are presented as percentage of wild-type level. *P 0.05, **P 0.001. Error bars denote s.e.m.
The R2-P2 method for high-throughput robotic magnetic sample preparation for phosphoproteomic profiling.Figure 1KingFisherTM Flex configuration for R2-P2. The robotic configuration allows for loading of 8 different 96-well plates. Each plate can be rotated into position under a 96-pin magnetic head that drops down inside the 96-well plate to release, bind or agitate the magnetic microspheres in solution. In the first robotic run proteins are captured from lysates by carboxylated magnetic beads, purified, and eluted by digestion at 37°C. Eluted peptides are dried down and can be resuspended for total proteome analysis by LC-MS/MS and/or for automatic phosphopeptide enrichment. Phosphopeptides are enriched using a second robotic run on the KingFisherTM Flex, using Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic microspheres, and analyzed by LC-MS/MS to obtain the phosphoproteome. 200 µg of yeast protein extract in different buffer compositions was processed by R2-P2 followed by DDA-MS peptide analysis. Sample preparation was performed in triplicate. Mean count of unique phosphopeptides are shown for the different conditions (n=3).   Fraction of phosphopeptides with 0, 1 or 2 missed cleavage sites. The mean is displayed (n=3).   Boxplot depicting phosphopeptide MS1 signal intensity distributions.   Boxplot depicting coefficients of variation (CVs) distributions for phosphopeptide MS1 signal intensities (n=3).
Figure 5(A) Consort diagram of the collected and analyzed FFPE tumor samples from patients with HBOC syndrome following the scoring criteria for the RAD51 assay. (B) Percentage of geminin-positive, RAD51 or γH2AX nuclear foci-containing cells in FFPE tumor samples from patients with HBOC syndrome. The box underneath summarizes the patient's young onset (<35y: <35 years), her family history (FH, purple box for PALB2-related tumors) and the presence of BRCA1 nuclear foci in the analyzed tumor samples. (C) Immunofluorescence staining of γH2AX, BRCA1 and RAD51 foci in three representative FFPE tumors from patients: one RAD51- and BRCA1-positive tumor (Pt03), one RAD51- and BRCA1-negative tumor (Pt11) and one PALB2-related tumor, with BRCA1 but not RAD51 nuclear foci (Pt20.1). Scale bars: 10µm.
Figure 1(A) Schematic diagram of SARS-CoV-2 Spike protein and the Spike constructs, Spike 1-1273 (full length), 1-674 (S1), and 319-591 (RBD-SD1) used in this study. The arrows indicate the cleavage sites of furin and TMPRSS2. (B) Western blot analysis of SARS-CoV-2 Spike 1-1273, 1-674, and 319-591 expression in HEK293T cells. The black arrows indicate the location of the induced target proteins. (C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining. (D) Recognition of the ACE2-Fc and Spike S1-Fc by anti-human IgG Fc antibody in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (E, F) Deglycosylation of ACE2-Fc and Spike 1-674-Fc by PNGase F. PNGase F digested ACE2-Fc (500 ng) and Spike 1-674-Fc (500 ng) were subjected to Coomassie Brilliant Blue staining (E) and Western blot analysis by anti-human IgG Fc antibody (F).
SENP3 is associated with PELP1, TEX10 and WDR18.Figure 1(A, B) Flag‐SENP3 or HA‐PELP1 was expressed in HeLa cells as indicated. Proteins were captured on Flag‐ or HA‐beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated. (C) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti‐SENP3 antibody. (D-F) Flag‐ or HA‐tagged proteins were expressed in HeLa cells, captured on Flag‐ or HA‐beads and subjected to immunoblotting as indicated. In all experiments, the inputs represent 2.5% of the total cell lysate.
Fig.Figure 3(A-C) DA level (pictograms per head) in males of the indicated genotypes. n = 3, 3, 3 (A); n = 4, 5, 3, 3 (B); n = 3, 3, 3 (C) times (100 heads per genotype). (D) Posterior confocal sections of UAS-mCD8-GFP; Ddc-Gal4 adult male brain in combination with UAS-LacZ or UAS-Myc.RNAi-1 stained with antibody to nc82 (pan-neuropil marker; magenta). Scale bars represent 50 μm. (E) Quantification of GFP and nc82 fluorescence intensity in GFP positive somata area (n = 10 heads per genotype). Statistical significance was compared with relative control. (F, G) Western blot of proteins extracted from adult heads of indicated genotypes. Statistical analysis showing the protein level of GFP (F) or Ddc (G) to that of β-actin in Western blot.
The limited enzyme supply defines yeast metabolic strategies at high energy demand in glucose-limited cultures.Figure 3Figure 3: The limited enzyme supply defines yeast metabolic strategies at high energy demand in glucose-limited cultures. The light blue areas denote the dual limitation (Janusian) region. (A) Chemostat aerobic data and model simulations at increasing dilution rate. (B) Predicted top 6 pathways used in terms of mass at increasing dilution rate in aerobic conditions. (C) Reduced flux variability compared with purely metabolic GEM simulations at increasing dilution rate in aerobic conditions. (D) Chemostat anaerobic data and model simulations at increasing dilution rate. (E) The model chooses to ferment when energy requirements for non-growth maintenance are high.
Fig.Figure 2A. The distance traveled (left panel), the speed (middle panel) and the time spent in the center region (right panel) during a 5 min open field test was similar amongst 3 months old CamkIIδc TG (n = 16) and control mice (n = 13). Unpaired t-test, two-tailed. B. The time to enter the escape hole during training sessions of the Barnes maze test is impaired in old CamkIIδc TG (n = 16) and control mice (n = 13; two-way ANOVA, *p<0.05). C. Plots showing the different search strategies of CamkIIδc TG (n = 16) and control mice (n = 13) across training trials. Each strategy is labeled with a unique color. D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). Data information: Bars and error bar indicates mean ± SEM.
Figure 4A. Typical fitted photon counting histograms of GFP and GFP-GFP in live yeast cells. B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin. C. PCH analysis of strain yAM085 (SMC3-GFP) throughout the cell cycle. D. PCH analysis of strains yAM953 (SCC1-GFP SMC3-GFP) and yAP941 (SCC1-GFP SCC3-GFP) containing two GFP tagged subunits. Data shown as boxplot (+data overlap) with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) from at least three independent experiments.
CHK1-inhibition kills primary murine HSPCs.Figure 2LSK (Lin- Sca1+ cKit+) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13.5 or the bone marrow of adult mice and treated for 48h with PF-477736 [1.5 μM]. Survival was assessed using AnnexinV-staining and flow cytometry. Bars represent means ± S.E.M. from n=6 wild type and n=3 Vav-BCL2 fetal liver LSK cells (left) and n=3 bone marrow LSK cells (right). WT and Vav-BCL2 derived E14.5 total fetal liver cells (B) were treated for 72h (B) with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype). WT and Vav-BCL2 derived E14.5 total bone marrow cells (C) were treated for up to 48h with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).
Figure 5(A) Cell viability test of DMKG (2 mM)-treated CRISPR-generated cystinotic cells (CTNS‑/-) upon pre-treatment with cysteamine (100 µM), bicalutamide (35 µM), and a combination of cysteamine and bicalutamide (100 µM and 35 µM, respectively). Results are shown relative to the starved condition (n = 6). (B) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in DMKG (2 mM)-treated CTNSWT and CTNS-/- cells upon pre-treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 3). (C) Relative reactive oxygen species (ROS/mg protein) production in DMKG-treated CTNSWT and CTNS‑/- cells upon pre-treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 3). (D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images). (F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6). (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).
(A) Relative expressions of RIPK1 and RIPK3 were analyzed by western blotting in Socs6-/- BMDMs stimulated with Mtb for 0-24 hours.Figure 6(A) Relative expressions of RIPK1 and RIPK3 were analyzed by western blotting in Socs6-/- BMDMs stimulated with Mtb for 0-24 hours. Representative blots from n = 3 biological replicates are shown. (B) Socs6-/- BMDMs were stimulated with Mtb for 0-24 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblots. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown. (C) Socs6-/- BMDMs were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant for 24 hours. Afterwards, transfected cells were stimulated with Mtb for 12 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown. (D-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.
Figure 6A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um. B. Quantification of the mean fluorescence intensity of samples in (A). Each dot represents a cell. C. Schematic depicting the infection and interferon addition to evaluate ISG15 induction following SARS-CoV-2 and astrovirus infection. D. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment RNA was harvested and the induction of ISG15 was analyzed by q-RT-PCR and normalized to the housekeeping gene TBP. N=3 biological replicates. Error bar indicates standard deviation. Statistics were determined by unpaired t-test. E. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were fixed and stained for either the SARS-CoV-2 N protein or for human astrovirus 1 (HAstV1) capsid protein. N=3 biological replicates. Representative image is shown. Scale bar=100 um. F. Schematic depicting the T84 MX-1 mcherry expressing cells and their response to IFN or SARS-CoV-2 infection. G. T84 MX-1 mcherry were infected with SARS-CoV-2 at an MOI=3 or MOI=1. 24 hpi cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment cells were fixed and stained for SARS-CoV-2 N protein. The number of MX-1 positive, SARS-CoV-2 positive and double positive cells was quantified. N=3 biological replicates were performed. Error bar indicates standard deviation.
Oligo#5 does not influence RNA editing of the serotonin 2C receptor and acts directly on pre-mRNA.Figure 3A. The RT-PCR product generated from cells treated with oligo#5 was directly sequenced using Sanger sequencing method. The fluorescent peaks after capillary electrophoresis are shown. Letters in gray boxes indicate the editing sites (also shown in Figure 1C). B. Sequence of the weak control band, generated without oligo#5 (Figure 1C, lane (-)). C. Structure of the in vitro minigene: The construct is schematically shown on top and consists of exon Va, Vb, 115 nt of the downstream intron and a MINX exon with 75 nt (dashed line) of the upstream intron. A striped box indicates the MINX exon. D. In vitro splicing assay: Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract (NE) under splicing conditions for two hours at 30 ºC. The structures of the in vitro splicing products are shown on the right. LI and LII are two lariats formed. Numbers indicate the expected product lengths.E. Quantification of selected bands from four independent experiments. The percentage of each form in the total products is shown. The differences of splice products (Va+b-MINX) is significant, p=0.0055; n=3.
Atg16L1(1–230) localizes to aberrant membranes in a FIP200‐independent manner.Figure 5(A) Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and subjected to immunofluorescence microscopy using anti‐Atg16L1 antibody. Note that full‐length Atg16L1 and Atg16L1(1-230), but not Atg16L1Δ(230-300), showed punctate structures under starvation conditions (inset). (B) FIP200 KO MEFs stably expressing either CFP-Atg16L1(1-588), CFP-Atg16L1(1-230) or CFP-Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP antibody. Arrowheads indicate the perinuclear localization of Atg16L1(1-230). (C, D) FIP200 KO MEFs stably expressing CFP-Atg16L1(1-230) were cultured in regular DMEM. Scale bar, 10 μm, and 2 μm in inset. (E) Hypothetical model of membrane targeting of Atg16L1. Interaction of Atg16L1 with FIP200 leads to proper targeting of this complex to the autophagosome formation site (middle). Without FIP200, puncta formation of Atg16L1 is impaired probably owing to a self‐inhibitory role of the C‐terminal WD‐repeat domain (left). If the WD‐repeat domain is deleted, the N‐terminal half of Atg16L1 localizes to aberrant membranes as well as the autophagosome formation site (right). KO, knockout; MEFs, mouse embryonic fibroblasts.
a, Co-localization of mCherry–TRPML1 and EGFP–LAMP1 at the membrane of an isolated enlarged LEL (see Methods).figf3a, Co-localization of mCherry-TRPML1 and EGFP-LAMP1 at the membrane of an isolated enlarged LEL (see Methods). The patch pipette was filled with red rhodamine B dye (shown in blue for the purpose of illustration). c, Lysosomal ITRPML1Va. Switching from lysosome-attached to (lysosome) luminal-side-out configuration significantly reduced the amplitude of the current. The luminal-side-out patch was exposed to the Tyrode's solution. A Cs+-based solution (147 mM Cs-methanesulphonate (Cs-MSA)) was used as a pipette solution for both configurations. d, NMDG+-impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations (nominal divalent-free). e, Lowering pH potentiated lysosomal ITRPML1Va. f, IFe/TRPML1Va induced by 30 mM and 105 mM Fe2+. g, Whole-lysosome current in an enlarged lysosome expressing wild-type TRPML1. The pipette (lumen) solution contained nominal divalent-free Tyrode solution. A Cs+-based bath solution (147 mM Cs-MSA) was used. h, Whole-lysosome ITRPML1Va. i, Whole-lysosome IFe/TRPML1. The pipette (lumen) solution contained 105 mM Fe2+ (pH 4.6).