EMBO/sd-character-level-ner huggingface dataset:
ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.
(J) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in Achilles tendon enthesis compartment. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage
(B) Fusion events result in the spread of mtPA‐GFP across the mitochondrial population, leading to its dilution and a decay in GFP FI value (n=16).
(A) H1299 neo and HA‐BCL‐2b5 cells treated with either CTRL or NAF‐1 shRNA were loaded with Fura‐2AM, and ER Ca2+ stores were measured as the difference in cytoplasmic Ca2+ concentration before and after addition of TG (2 μM). Shown are representative traces of Fura‐2AM fluorescence measured at 340/380 nm excitation wavelength ratio at 510 nm wavelength emission. Arrow indicates time at which TG was added, delta values indicate TG‐sensitive ER Ca2+ stores.
(g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments.
(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.
(b) IHC for Trim28 in the adult cortex revealed that the protein was lost in cells exposed to Cre-activity during brain development (GFP+ cells). Scale bars: low magnification 75 μm, high magnification 20μm.
B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization.
Bar graph showing the relative levels of free/total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.
E-G. PEO patient (P1) muscle before diet:E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µmF. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).G. Enlargement of the area marked in F; mitochondria with paracrystalline "parking lot" inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µmH-J. PEO patient (P1) muscle after mAD (H-J):H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µmI. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µmK. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.
A P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice.
E. Diagram showing the experimental design, including procedure of MOS mRNAs injection and oocyte culture. GV oocytes were overexpressed via microinjection with wild-type MOS or MOS variant mRNAs combined with mCherry mRNAs, followed by culture for 4 h in medium containing 2.5 µM milrinone.
(C Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.
E, F Modulation of Anp and Bnp mRNA levels by Ad-LXRα-induced O-GlcNAc signaling. Gene expression as determined by RT-PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.02.
B) Microtubules are tightly associated with the nucleus. Z stacks of representative CD38- and CD33+ cells are shown. Microtubules are shown in green, nucleus envelope (Sun2 immuno-staining) in magenta and chromatin in blue. Scale bar 5 µm.
Differentially abundant taxa are displayed for (B) infantsat per visit (FDR adjusted p-value < 0.05).
Quantification of NRP1 protein expression in LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl BMDM; n= 6.
(I) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 60 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N =3.
A. STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set. Pearson correlation between STAT3 and PDK4 is shown. Kaplan-Meier plot shows stratified subgroups. P-values were estimated by Log-rank test and adjusted with Benjamini-Hochberg method. Hi = high, lo = low.
c. Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell-cell fusion.
A-F. Immunofluorescence images of midgut sections from the R4 region in Drosophila carrying esgts-GAL4-driven expression of CAT cDNA (A), CAT cDNA with AD4 administration (B), CAT cDNA with ALA administration (C), Keap1 RNAi (D), Keap1 RNAi with AD4 administration (E), or Keap1 RNAi with ALA administration (F). esg-GFP (green) represents ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. G. Quantification of the number of esg-GFP+ cells, Dl+ cells of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. n is indicated. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, 19, 18, 19, 20, 18, 18, 19, and 19. H. Quantification of the number of pH3+ cells and luciferase activity of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, and 19. I- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.
Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP-mutants at 15 hpi, 2 dpi or 6 dpi (n=5 per group). The concentrations of 36 immune mediators were quantified using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Each color represents the relative concentration of a particular analyte. Blue and red indicates low and high concentration, respectively.
G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars).
Length distribution of total small RNAs from BmN4 cells overexpressed with AcGFP, mCherry-Siwi or mCherry-Siwi-D670A. Two peaks (20-nt and 27-nt) correspond to siRNA/miRNA and piRNA respectively.
(E) Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 following expression of SOX9-WT or SOX9-T236/240A. Total RNA were collected 24 hours following doxycycline treatment, from which cDNA were generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2-way ANOVA with Bonferroni's post-test.
(B) Electrostatic surface of YqgPNTD structure reveals highly negatively charged areas. Left view is oriented as the left view in panel (A).
(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous Rab5 (in green) and EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.
D SHP1IP in the presence and absence of SHP1 tail phosphorylation. SHP1 was immunoprecipitated from J.CaM1.6LCK‐Tet cells. Where indicated, LCK expression was induced with doxycycline prior to the experiment or Src‐kinase inhibitor PP2 was used to abolish residual SHP1‐pY564. The upper panel of immunoblots shows the effect of LCK activity on SHP1‐pY564 levels. The red arrowhead indicates residual pY564. Isolated protein complexes are shown in the lower panels.
D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs.
Confocal microscopy images of whole-mount preparations of cochlear sensory epithelia from Clrn2clarinet/+ (C), Clrn1-/- (D), and Clrn2clarinet/clarinet (E) P6 mice immunostained for stereocilin (green) and actin (red). Unlike the fragmented immunostaining in Clrn1-/- mice (mirroring the fragmentation of the hair bundle), stereocilin immunostaining in Clrn2clarinet/clarinet mice reflects the normally V-shaped bundles of OHCs, similar to Clrn2clarinet/+ OHCs.
(G) Increased polyubiquitination of SCAP in RNF5-transfected HEK293 cells. 293T cells were transiently transfected HA-tagged RNF5 for 18 hours followed by MG-132 treatment for 3 hours. Arrow indicated HA-tagged RNF5.
qRT-PCR measurement of of Ifnb1, Il6 and Il1b mRNA transcripts in mouse peritoneal macrophages left unstimulated or stimulated with LPS (0.1 μg/mL) (A-C) or transfected with Poly(I:C) (1μg/mL) The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. ns, not significant.
D, Model of the intact ColE9 translocon generated after docking and rigid-body refinement of individual structures of ColE9 residues 85-580 (PDB ID 5EW5), OmpF (3K19), BtuB (PDB ID 2YSU), and TolB (PBD ID 4JML).
D) Comparison of polygon distribution for the 10% smallest cells from BCA (green bar) and Control Simulation (grey bar) with the sick cells from BNA (light blue bar) and Atrophy Simulation (grey cells from (B), purple bar).
C, Whole-cell lysates from the indicated time points were subjected to immunoblotting with the indicated antibodies.
B. Frequency of ISV ectopic branching occurring at the HM (n=4 experiments; *P<0.05; one-way ANOVA with Bonferroni's correction). +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (B).
(C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD.
(A) Wild‐type (YW5‐1B), apg10‐1 mutant (MT91‐4‐2) and Δapg10 (TFD10‐L1) cells were incubated in SD(−N) medium containing 1 mM PMSF at 30°C. After incubation for 6 h, cells were observed under light microscopy (Nomarski images).
IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.
Principal component analysis (PCA) of brain samples based on the abundance of all proteins identified by label-free mass spectrometry. The smaller dots represent individual samples and the larger dots the centroids of each age-matched group. Ellipses represent 95% confidence intervals. The percentage of variance explained by the first two PC axes is reported in the axis titles.
F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars). Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).
F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.
Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.
(E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester.
(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3) P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (
(C) Viremia progression in virus-infected mice was monitored over 2 weeks (n=11 per group). Viremia is detected with CHIKV nsP1 probe via qRT-PCR. Statistical
(D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
C, A diagram showing the BIR/RMD model when a single-ended DSB is generated after replication fork collapse.
Cortical thickness measured adjacent to HPC, from CTX surface to corpus callosum, was decreased in rTg4510 mice by ≈25% compared to WT mice. rTg4510-Mapt0/0 showed no CTX thinning compared to Mapt0/0 or WT mcie. Mean±SEM, n=3 mice/group.
XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments
A) Left panel: overlay of the models resulting from the introduction of Arg and Asp into the 2M5N zipper structure, with the identifier indicated for each strand. Strands E and F are highlighted in red. Right panel: the same structure turned 90° about both the x- and y-axes. Arrows in (a) indicate the capacities of Arg and Asp to distance their charged moieties. (b) shows an example of planar Arg stacking.
(e) Interaction of endogenous p62 with Keap1. Lysates prepared from the human hepatocellular carcinoma cell line Huh-1 were immunoprecipitated with anti-p62 antibody or anti-Flag antibody (negative control) followed by immunoblotting with antibodies against p62 and Keap1. The bands corresponding to endogenous p62, Keap1 and IgG heavy chain (IgG H.C.) are indicated. The data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.
C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.
Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.
(F-G) EM from Dmef2-Gal4, UAS-whitei larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2-Gal4, UAS-whitei larva starved on low-nutrient food for 6 h. Arrow points to double membrane.
FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.
D. Model for domain movements in the AAA ring of dynein during ATP hydrolysis at AAA1. The AAA ring can be divided into two halves that are connected by two springs. Upon ATP binding / hydrolysis, the gap between AAA1/ AAA2 closes and moves AAA6 / AAA5 which in turn pulls on AAA4 so that the gap between AAA4 / AAA5 closes as well. In addition, this conformational change will pull the buttress and therewith change the stalk registry.
(A) The purity of the isolated lysosomes. Total cellular homogenates and lysosomal fraction (30 µg of proteins) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Actin was shown as a marker of cytoplasm. Hsp60 was shown as a marker of mitochondrial. LAMP-2A and Hsc70 were shown as markers of lysosomes.
(D) Average number, normalized on cell size, quantified by image (as in a) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are the average ± SEM of two independent experiments for a total 830 early endosomes in 13 shCTL/GFP cells (64 ± 6 endosomes per cell), 719 early endosomes in 10 shSTIM1/GFP cells (60 ± 6 endosomes per cell), 820 early endosomes in 12 shSTIM1/GFP-STIM1 WT cells (68 ± 6 endosomes per cell) and 400 early endosomes in 12 shSTIM1/GFP-STIM1 NN cells (33 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,01 **, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p> 0.05 not significant (ns), for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,001 *** and for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,001 ***.
A) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous, CAF1-knockdown-induced (through p60 or p150 KD) 2-cell-like cells. Fold-changes relative to ESCs were calculated based on bulk RNA-seq data
(A) The efficiency of knockdown and overexpression of Hsp. HeLa cells were transiently transfected with Hsp70 plasmid or Hsp70 siRNA for 72 h. Lysates were subjected to Western blot analysis with anti-Hsp70 antibody. Values are the mean±SE of three independent experiments. *P<0.05, **P<0.01 (vs control).
Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments.
C,D, Mean ChIP-seq signal MED1 and MED16 surrounding gene promoters from gene groups defined in Fig 4.
A. Two routes of MT polymerization in the presence of DCX are considered: direct nucleation, involving stabilization of early tubulin oligomers by DCX; stabilization, involving DCX binding only between straight PFs of a complete MT lattice. Question marks, unknown mode of DCX binding to tubulin/MT.
(D) Kaplan Meier plots for progression-free survival in RCC patients stratified into groups of high or low levels of APELIN and VEGF. Cut-off levels were set by the median. Serum was analysed 3-5 months after the start date of sunitinib treatment. *P<0.05, **P<0.01; Log rank test.
Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.
ZO-1 quantification of IF images performed using the Fiji software and bar-plotted as normalized integrated densities per cell (grey dots) with bars corresponding to Mean ± Standard Error of the Mean (SEM) for at least 11 high-field units of at least 100 cells; * P-value < 0.05 (Student's t-test, N=11).
Reproducibility of the S-scores using the two batches used in the screening. Reproducibility of the S-scores using genes having multiple independent colonies plated in the screening.
(A and B) Lactate production assay in BT549 (A) and MDA-MB-231 (B) cells with Luciferase or GLUT1 knockdown.
(D) Two-color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labelled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments.