EMBO/soda-nel huggingface dataset:
Regulating retrotransposon activity through the use of alternative transcription start sites (2016)
Figure 8

Figure 8. Tf2 retromobility assay

A) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D (PP3) and fft2Dfft3D (PP4) mutant strains with new integrations of Tf2-12-neoAI. After 3 days at 32o C. B) A Table showing the Tf2 transposition frequencies and rates calculated with fluctuation tests using FALCOR. C) Southern blot probed for neoA fft3D (PP3) and fft2Dfft3D mutant strains (PP4) and derived G418 resistant subclones.
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 1

Figure 1. COUP-TFII expression is increased in human fibrotic kidneys and lungs, and human kidney organoids. (A) COUP-TFII mRNA levels in human kidney tissues of control (n=8) and CKD (n=53) subjects from GSE66494. ***p<0.001 by t-test. (B) COUP-TFII expression (green) is increased in human kidneys from patients with thrombotic microangiopathy (TMA) and diabetic nephropathy (DN) (n=2). Quantification by confocal micrographs in 400x hpf. ****p<0.0001 by one-way ANOVA; mean ± SD. The majority of these cells were localized in the interstitial region. More than 50% COUP-TFII (+) cells (green) are co-localized with expanded αSMA-positive (red) areas of fibrosis. Pearson correlation coefficient (PCC) = 0.56 ± 0.1 for patient#1 and 0.65 ± 0.13 for patient#2. (C) COUP-TFII expression (green) is increased in lungs from patients with IPF (n=2) by immunofluorescence. Quantification by confocal micrographs in 400x hpf. **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. (D) Human kidney organoids were treated with IL-1β (10ng/ml) for 96 hours. Immunofluorescence reveals significantly increased COUP-TFII (green) and αSMA (red) expression in IL-1β treated organoids, compared to non-treated organoids. Quantification by confocal micrographs in 200x hpf. **p<0.01; ***p<0.001 by t test; mean ± SD. (E) COUP-TFII expression increased significantly in organoids treated with TGFβ1 (10ng/ml), cobalt chloride (100 μM), IL-1β (10ng/ml), but not PDGF (50ng/ml). Quantification by confocal micrographs in 400x hpf. *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. The higher magnification of the insert from IL-1β φησwed co-localization of COUP-TFII (green) and αSMA (red) staining.
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 2

Figure 2. COUP-TFII protein is expressed in pericytes/fibroblasts, both in non-injured and injured mouse kidneys. (A) COUP-TFII+ cells (green) express pericyte/fibroblast marker (PDGFRβ) but not the endothelial cell marker (CD31). (B) Strategy of genetically labeling Foxd1-derived stromal cells in non-injured mouse kidney. (C) Majority COUP-TFII+ cells (green) were also positive for Foxd1 (tdTomato-labeled as red) in the non-injured mice kidney (n=2). Quantification by confocal micrographs in 400x hpf. (D) COUP-TFII was enriched most in pericytes/fibroblasts in injured kidney, both in the human (Diabetic kidneys, Fig 2d top) and mouse model (UUO day 14, Fig 2d bottom) as analyzed from single cell sequencing database (http://humphreyslab.com/SingleCell/).
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 3

Figure 3. COUP-TFII expression is increased in the injured mouse kidneys during the development of kidney fibrosis. (A) COUP-TFII expression is significantly increased and co-localized with αSMA+ cells at day 10 of UUO and day 14 of UIRI in mice (n=3 animals per group). Quantification by confocal micrographs in 200x hpf. *p<0.05 **p<0.01 by paired t test; mean ± SD. (B) In a time course of UUO, COUP-TFII expression increased as early as day 2 and preceded the increased expression of αSMA (n=2 animals per time point). Quantification by confocal micrographs in 200x hpf. *p<0.05; ***p<0.001 by two-way ANOVA test; mean ± SD. (C) In the UUO model, COUP-TFII positive cells do not stain with markers of inflammatory cells (T cell marker CD3, neutrophil marker LyG6, and macrophage marker F4/80) (n=3 animals). (D) Using a fate tracing of Gli1+ cells in the UUO model, (E&F) Gli1-tdTomato+ cells expanded primarily in the outer medullary region. In contrast, COUP-TFII+ cells are distributed both in cortex and medulla. A subset of COUP-TFII+ cells (green) overlap with genetically labeled Gli1+ pericyte/perivascular cells (red) in UUO injury model (n=3 animals per group). CTL: contralateral kidney. Quantification by confocal micrographs in 200x hpf.
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 4

Figure 4. Genetic ablation of COUP-TFII in adult mice attenuates injury-induced kidney fibrosis. (A) Strategy of conditional knockdown of COUP-TFII in adult mice. A LacZ knock-in allele is inserted into the genomic COUP-TFII locus after the second LoxP site. (B) Activation of Cre recombinase by tamoxifen (TAM) results in COUP-TFII deletion and the expression of LacZ reporter (detected by immunostaining of β-galactosidase (β-Gal, red) in the UUO mice model (n=2). Quantification by confocal micrographs in 200x hpf. **p<0.01; ***p<0.001 by paired t test; mean ± SD. (C) Using heterozygous mice (F/+; Cre/+), β-Gal+ cells (red) increased after TAM injection in UUO kidneys, and co-localize with COUP-TFII+ cells (green) (n=3). (D) COUP-TFII+ cells decrease significantly in the UUO kidney in TAM-treated homozygous (F/F;Cre/+) mice (KO group, n=6) compared to wild-type littermates (WT group, n=4). Expression of αSMA (red) and collagen1 (yellow) are also markedly reduced. Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group. Quantification by confocal micrographs in 200x hpf. **p<0.01 by unpaired t test, mean ± SD.
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 5

Figure 5. Cellular knockout of COUP-TFII in vitro in C3H/10T1/2 cells decreases cell proliferation and suppresses TGFβ-induced αSMA and collagen1 expression. In contrast, overexpression of COUP-TFII alone increases collagen1 production. (A) Verification of COUP-TFII loss- and gain-of-function cell lines generated by CRISPR-Cas9 and an inducible lentiviral construct in pericyte-like cell line C3H/10T1/2 in vitro (n=6), ****p<0.0001 by paired t-test. (B&C) COUP-TFII KO had no effect on cell viability, although decreased proliferation rate compared to naïve C3H/10T1/2 cells (WT) (n=6), ****p<0.0001 by two-way ANOVA. (D) COUP-TFII-OE cells, in the absence of TGFβ1 stimulation, displayed an elongated fibroblast shape with increased αSMA expression, similar to WT cells treated with TGFβ1. Quantification by confocal micrographs in 400x hpf, *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD. (E) Overexpression of COUP-TFII alone without TGFβ1 induces collagen1 production (n=3). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 6

Figure 6. COUP-TFII enhances TGFβ1-induced glycolysis during myofibroblast differentiation, which is essential for collagen production in vitro. (A&B) Non-biased proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells revealed that COUP-TFII promotes the expression of proteins enriched in metabolic process critical for myofibroblasts, in particular, suppression of FAO and enhancement of glycolysis. Top pathways ranked by NES (gene set enrichment with FDR < 0.05) altered in COUP-TFII-OE compared to naïve C3H10T1/2 cells (n=2). (C) TGFβ1 significantly increased more lactate production by COUP-TFII-OE (OE) cells when compared to WT or COUP-TFII-KO (KO) cells treated with TGFβ1. Interestingly, overexpression of COUP-TFII alone (without TGFβ1) significantly increased lactate production compared to WT (n=3). **p<0.01, ****p<0.0001 by one-way ANOVA, mean ± SD. (D) WT, COUP-TFII overexpression (OE) and COUP-TFII knockout (KO) cells were seeded in Seahorse XF-24 cell culture microplates. The cells were rendered quiescent in 0.5% FBS DMEM overnight and then treated with or without 10 ng/ml TGFβ1 for 24 hours. All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate, followed by sequential treatments with glucose (10mM), oligomycin (5μg/ml) and 2-DG (50mM). Real-time extracellular acidification rate (ECAR) was recorded as the baseline (before glucose), the rate of glycolysis (after glucose), glycolytic capacity (after oligomycin), and glycolytic reserve (after 2-DG) (n = 10). (E) COUP-TFII OE significantly increased, while COUP-TFII KO decreased, in the baseline, rate of glycolysis, glycolytic capacity, and glycolytic reserve when compared to WT cells under TGFβ1 stimulation. Without TGFβ1 treatment, there is no difference on the baseline and glycolysis among WT, OE, and KO cells. COUP-TFII KO significantly decreased glycolytic capacity and reserve compared to WT, even without TGFβ1 stimulation (n=10), *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. (F) 2-deoxyglucose (2-DG) treatment inhibits TGFβ1-induced glycolysis (left) and fibrosis (right) in COUP-TFII-OE cells by qRT-PCR (n=6). **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. We confirmed that 2-DG suppressed TGFβ1-induced proteins of αSMA and ColA1 in COUP-TFII OE cells without change the protein of COUP-TFII by western blot (n=3). **p<0.01 by one-way ANOVA.
Orphan nuclear receptor COUP-TFII enhances myofibroblasts glycolysis leading to kidney fibrosis (2021)
Figure 7

Figure 7. Overexpressing COUP-TFII resulted in decreased transcription of PGC1α and PDK4, and knockout of COUP-TFII increased PGC1α and PDK4 in vitro. Overexpression of PGC1a, however, is not sufficient to suppress TGFβ1-induced glycolysis and COUP-TFII induced myofibroblast differentiation. (A) Volcano plot of all detected proteins from proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells. PDK4 expression is significantly decreased in OE cells (n=2). (B) Overexpression of COUP-TFII inhibited, and knockout of COUP-TFII increased, mRNA of PGC1α and PDK4 (n=6). ***p<0.001, ****p<0.0001 by one-way ANOVA, mean ± SD. (C) Chip-qPCR analysis on C3H10T1/2 cells in vitro revealed binding of COUP-TFII on the promoter of PGC1α, but not αSMA or PDK4 (n=2). **p <0.01, mean ± SD. (D) Overexpression of PGC1α was achieved through adenovirus transduction in C3H/10T1/2 cells (WT) and confirmed by qRT-PCR (n=6). Noted TGFβ1 significantly decreased PGC1α expression. ***p<0.001, ****p<0.0001 by one-way ANOVA, mean ± SD. (E) PGC1α OE significantly increased expression of Cpt1 and PDK4, both are involved in key steps of FAO. However, overexpression of PGC1α did not abrogate the TGFβ1 up-regulated genes in glycolysis and αSMA (n=6). *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. (F) PGC1α OE did not decrease αSMA protein neither without nor with TGFβ1 in COUP-TFII OE cells by WB (n=3). (G) Schematic model of the role of COUP-TFII in metabolic reprogramming during myofibroblast differentiation and fibrosis formation after injury.
Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis (2007)
figf1

a, Internalization of beads with PAM3CSK, LPS, or Alexa-Fluor-555-labelled BSA (Red-BSA; yellow arrows) and association with GFP–LC3 in RAW cells was followed by time-lapse video for 3 h, 1 frame/2 min (representative frames are shown). b, GFP–LC3 in RAW cells translocates to phagosomes containing zymosan (red), assessed as in a. c, Time course of translocation of GFP–LC3 to a phagosome containing zymosan (red) (see Supplementary Movie 2). Time (minutes) is indicated in the top left of each panel. d, LC3 translocation to the phagosome is induced by TLR signalling. GFP–LC3 was transiently transfected into wild-type or Tlr2-/- macrophages, which were exposed to zymosan for 45 min. e, The percentage of phagosomes (n ≥ 50 per group) with GFP–LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).
Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis (2007)
figf3

a, RAW cells expressing GFP–LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP–LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown). c, Bone-marrow-derived macrophages from wild-type and Atg7-/- mice were transiently transfected with GFP–LC3. After 24 h macrophages were fed with zymosan (red) for 1 h. Confocal images of the same cell before and after zymosan engulfment are shown. d, The percentage of GFP–LC3-associated phagosomes (n ≥ 50 per group) was assessed after 1.5 h in wild-type and Atg7-/- macrophages (represented as means and ranges; three mice per group). e, Ly294002 (Ly29, 10 &mgr;M) was added 10 min before zymosan, and wortmannin was added after engulfment but before GFP–LC3 translocation in RAW-GFP–LC3 cells. Representative images are shown. f, Percentage of GFP–LC3-associated phagosomes (n ≥ 50 per group) with P(I)3K inhibitors assessed by time-lapse video (means and ranges are shown; three independent 3 h videos). g, RAW cells expressing GFP–LC3 and the PX domain of p40(phox) fused to mCherry (red) were fed with zymosan (yellow arrows) or with beads (Supplementary Fig. 6). Internalization was followed at 1-min intervals (Supplementary Movie 5), representative images are shown. Single cells were followed for at least 2 h (n ≥ 20 per group) (Supplementary Movies 6, 7 and Supplementary Fig. 6). Mean ± s.d. duration of mCherry-PX translocation to the phagosome for beads (B) and zymosan (Z) are shown. h, Internalization of zymosan (yellow arrows) was followed at 1.5-min intervals for at least 2 h in RAW cells transiently transfected with GFP–BECN1. Representative images are shown. Translocation of GFP–BECN1 to the phagosome for beads (B) and zymosan (Z) was quantified (mean ± s.d.) using time-lapse movies (Supplementary Movie 8 and 9; n ≥ 20 cells per group). For g and h, time (in minutes) is indicated at the top left of each panel.
Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis (2007)
figf4

a, Internalization of control or PAM3CSK4-covered beads was followed in RAW-GFP–LC3 cells pre-loaded with lysotracker (red) by time-lapse video for 3 h. Time (in minutes) is indicated at the top left of each panel. b, RAW cells pre-loaded with lysotracker were exposed to control or PAM3CSK-covered beads for 6 h. Acidification of the beads (n ≥ 20 per group), measured as maximum lysotracker relative light units (RLU) on a horizontal cross-section of each bead represented in the x axis (mean ± s.d.; Wilcoxon–Mann–Whitney test, P  0.0005), is shown. c, d, Knockdown of ATG5 reduced zymosan-induced GFP–LC3 translocation and lysosomal fusion. e, Primary macrophages from wild-type (white bars) and Atg7-null mice (grey bars) were fed live yeast for 1 h and washed. Internalized yeast were extracted from the macrophages at 4 h and 8 h after re-plating. Means and ranges from six different measurements using two mice per group are shown. f, Initiation and duration of translocation of PX-mCherry to phagosomes (yellow bar), GFP–BECN1 translocation (blue bar), GFP–LC3 translocation (green bar) and Lysotracker recruitment (acidification) were determined during the first 2 h after internalization (10 cells per group, error bars indicate s.d.).
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 1

Figure 1. Localization of the identified GPHN mutation (NM_001024218; NP_001019389).

A Protein and genetic structure including the different gephyrin domains and genomic location of the gene with an indication of the different exons. The position of the mutation identified in this study is indicated on the protein level, affecting the E-domain and on the genomic level, affecting exon 12.

B Multiple alignment of gephyrin and homologous proteins from various species from different kingdoms of life (aligned using Clustal Omega).

C Co-crystal structure of gephyrin E-domain (blue) and bound GlyR ß-loop (red – PDB code 2FTS (Kim et al., 2006)). The mutated gephyrin Gly375 residue is shown in orange.
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 2

Figure 2. Expression of WT-gephyrin and gephyrin-G375D in primary neurons.

A Representative images of GFP-gephyrin and GFP-G375D, expressed in hippocampal neurons at 9 + 4 days in vitro. Inset shows higher magnification. Scale bar = 20 µm.

B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons. Number of gephyrin clusters in each dendrite is normalized to 20 µm segments.

C Representative segments of dendrites expressing GFP-gephyrin or GFP-G375D and immunostained with the presynaptic marker VGAT. Arrowheads show co-localization of gephyrin and VGAT clusters. Scale bar = 10 µm.

D Quantification of co-localization between gephyrin and VGAT clusters (gephyrin-VGAT 77.4±2.9 %, G375D-VGAT 30±4.4 %). 31 GFP-gephyrin and 30 GFP-G375D neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 3

Figure 3. Gephyrin-G375D acts dominant-negatively on postsynaptic clustering of wild type gephyrin in neurons.

A Representative dendritic segment of a neuron expressing GFP-G375D and 3B11-immunostained endogenous gephyrin (red).

B Quantification of density and (C) size of endogenous gephyrin clusters in neurons expressing GFP, GFP-G375D or non-transfected neighboring neurons. 20 GFP-G375 and non-transfected neurons and 15 GFP-expressing neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM and data were analyzed by t-test.
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 4

Figure 4. Gephyrin-G375D decreases cell-surface expression of GABAARs and impairs postsynaptic currents.

A Representative neurons expressing myc-tagged gephyrin or G375D and pHluorin-tagged GABAAR γ2. Surface-expressed γ2-subunits were immunolabelled with GFP-antibodies in red, and the pHluorin-fluorescence (total γ2 levels) are shown in green.

B Size and C fluorescence (fl.) intensity of surface-expressed γ2 clusters in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 myc-gephyrin and 21 myc-G375D neurons in B and 18 myc-gephyrin and myc-G375D neurons in C)

D Mean γ2-pHluorin fluorescence in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 and 18 myc-gephyrin and myc-G375D expressing neurons).

E Number of surface γ2 clusters on dendrites normalized to 20 µm segments (n = 18 myc-gephyrin and myc-G375D expressing neurons). At least three independent cultures were used for each experiment. Results are expressed as mean ± SEM; Data were analyzed by t-test.

F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP-gephyrin or GFP-G375D.

G mIPSC amplitudes from neurons expressing GFP-gephyrin or GFP-G375D. Numbers show analyzed cell-numbers. Data were analyzed by one-way ANOVA and post-hoc Tukey test.

H Frequency of the mIPSCs from neurons expressing GFP-gephyrin or GFP-G375D. The horizontal line within the boxes indicates the median frequency for the represented condition (bottom/top boundary indicates the 25th /75th percentile). Top and bottom error bars indicate the 90th and 10th percentiles, respectively. Analysis by Kruskal-Wallis test followed by post-hoc Mann-Whitney test.
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 5

Figure 5. Gephyrin-G375D is stably expressed and oligomerizes with WT-gephyrin.

A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin.

B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm.

C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units).

D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 6

Figure 6. Gephyrin-G375D has a significantly decreased binding-affinity towards GlyRs and GABAARs.

A Schematic representation of GABAAR α3-subunit with residues of the cytoplasmic loop that were synthesized and used as a biotinylated peptide for downstream applications. Gephyrin core-binding motif is shown in red.

B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.

C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.

D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.

E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.
Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy (2015)
Figure 7

Figure 7. Gephyrin-G375D is catalytically inactive and unable to synthesize Moco.

A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.

B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi & Schwarz, 2013)).

C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.