EMBO/soda-nel huggingface dataset:
Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation and protects from vascular damage in the retina (2016)
Figure 1

Figure 1. Inhibition of microglia/macrophage reactivity after intravitreal injection of polySia avDP20 in mice with retinal laser lesion.

A. Confocal images show Iba1-immunoreactive microglia and macrophages in RPE/choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Laser spots of vehicle-injected SIGLEC11 transgenic (tg) and wild type control animals revealed strong accumulation of activated microglia and macrophages, which was effectively decreased in polySia avDP20-injected SIGLEC11 tg animals and to a lower extent in wild type controls. Representative images out of at least three independent experiments are shown. Scale bar: 50µm.

B. Quantification of the average pixel intensity of Iba1-positive area in laser spots reflects accumulation of reactive microglia/macrophages on RPE/choroid whole mounts. In comparison to the accumulation of reactive microglia/macrophages in control animals, there is a reduced pixel intensity found in polySia avDP20-treated SIGLEC11 tg animals. Notably, treatment with a high polySia avDP20 dose (3 µg) also reduced accumulation of microglia/macrophages in laser spots of wild type animals. Data show mean +/- SD. * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA followed by Fisher’s LSD. WT PBS (n = 24 spots), WT 0.2 µg Sia (n = 37 spots), WT 3 µg Sia (n = 20 spots), SIGLEC11 tg PBS (n = 25 spots), SIGLEC11 tg 0.2 µg Sia (n = 35 spots), SIGLEC11 tg 3 µg Sia (n = 31 spots), WT PBS vs WT 3 µg Sia p = 0.0147, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0046, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0071, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0.0001.

C. Confocal images of retinal whole mounts show Iba1-immunoreactive microglial cells. Retinal microglia of polySia avDP20-treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS-vehicle treated mice. Interestingly, high polySia avDP20 dose (3 µg) also exerted a weak therapeutic effect in wild type animals. Representative images out of at least three independent experiments are shown. Scale bar: 20 µm.

D. Percentage of retina showing activated microglial cells within the laser spots was quantified. PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose (3 µg) also in wild type controls. Data show mean +/- SEM. * p< 0.05, ** p< 0.01, ***p<0.001, One-way ANOVA followed by Fisher’s LSD. WT PBS (n = 6 retinas), WT 0.2 µg Sia (n = 8 retinas), WT 3 µg Sia (n = 6 retinas), SIGLEC11 tg PBS (n = 8 retinas), SIGLEC11 tg 0.2 µg Sia (n = 7 retinas), SIGLEC11 tg 3 µg Sia (n = 6 retinas),WT PBS vs WT 3 µg Sia p = 0.0052, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0005, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0159, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.001.
Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation and protects from vascular damage in the retina (2016)
Figure 2

Figure 2. Reduced vascular leakage and membrane attack complex (MAC) formation after intravitreal application of polySia avDP20 in mice with retinal laser lesion.

A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.

B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher’s LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002.

C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.

D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher’s LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.
µLED-based optical cochlear implants for spectrally selective activation
of the auditory nerve (2020)
Figure 1

Figure 1: μLED-based oCI.

(A) Optically evoked auditory brainstem response in an AAV-CatCh-injected gerbil in response to a 1 ms laser pulse of ~35 mW (mean of 1000 stimulus presentations at 10 Hz repetition rate).

(B-D) Catch-eYFP-expressing SGNs in the apical cochlear turn identified by parvalbumin expression (B) and CatCh-eYFP (C). The inlay (D´) of the merged immunostaining (D) indicates the lack of CatCh-EYFP signal in inner hair cells (dashed white line). Scale bar: 20 µm.

(E, F) Scanning electron micrographs of a µLED (60 µm × 60 µm footprint) structured on a sapphire substrate showing the non-emitting p-contact side (E) and the emitting side of a µLED (F), transferred onto and embedded into an epoxy substrate. The GaN surface of the µLED has been roughened by KOH etching showing characteristic pyramidal structures (F, inset).

(G) Picture of an oCI carrying 16 individually addressable µLEDs with a pitch of 100 µm on a flexible substrate, µLEDs #5 and #13 (from the tip) are active.

(H) Radiant flux of individual µLEDs as a function of driving current. The thick line indicates the mean, error bars the standard deviation of the mean. Non-functional µLEDs (which did not emit light, 158 out of 560 µLEDs) have been excluded.

(I) oCI inserted into the cochlea (dashed black lines) via a cochleostomy in the basal cochlear turn (black, solid line). The round window niche is highlighted by a dashed white line, the round window by a solid white line. SA: stapedial artery.

(J) 3D X-ray tomography reconstruction of a 16-channel oCI implanted in a gerbil cochlea via the round window. Cables and μLEDs are marked in blue, the most apical µLED is indicated by the black arrowhead; the basilar membrane is marked in green. µLEDs have a size of 60×60 µm.
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 1

Figure 1. Stoichiometric binding of HIF-α and HIF-1β to chromatin in HKC-8 cells incubated in 0.5% atmospheric O2 for 16 hours. Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2.
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 2

Figure 2: Gene Set Enrichment Analysis (GSEA) showing hypoxic regulation of HIF-bound genes in (A) HKC-8, (B) RCC4 and (C) HepG2 cells. For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score).
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 3

Figure 3. The effect of graded hypoxia on HIF binding. HKC-8 cells were incubated in 21%, 3% or 0.5% ambient oxygen for 6 hours. (A) Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxia. (B) HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated. (E) Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines). (F) The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations.
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 4

Figure 4: The effect of duration of hypoxia on HIF binding. HKC-8 cells were incubated in 0.5% ambient oxygen for 6, 16 or 48 hours. (A) Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours. (B) HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepoint. (H) Frequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological difference. (F) The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints.
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 5

Figure 5: HIF-1α and HIF-2α do not compete for binding sites. The expression of either HIF-1α or HIF-2α was ablated in HKC-8 cells using CRISPR-Cas9. (A) Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines. (B) HIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell type. (C) HIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the other. Box-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel A. (E) The effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test). The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as above and of (G) HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test). The thick horizontal bars show the median, the boxes depict the interquartile range, and the whiskers represent the full range unless this is more than 2.5 times the interquartile range, in which case more extreme values are shown as individual points. ChIP-seq signal is averaged across two independent experiments.
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 6

Figure 6: Distinct binding distributions of HIF-1 and HIF-2 are conserved across cell types. Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types. (G) The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test).
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 7

Figure 7: Conservation of HIF-1α and HIF-2α specificity between cell types. Sites that bound (A) Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen. (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments.
Inherent DNA binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin (2018)
Figure 8

Figure 8: Transcriptional landscape at HIF-1 and HIF-2 binding sites in HepG2 cells. (A) Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia. (B) Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control. Expanded View Figure Legends
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 1

Figure 1. Identification of the β2AR Binding Region on Cav1.2.

(A) Schematic of the α11.2 subunit of Cav1.2 (top) and the α11.2-derived GST fusion proteins covering the C-terminus (bottom).

(B) Pull-Down of GST-tagged α11.2 segments (top immunoblot; IB) by immobilized MBP-β2AR C-terminus (residues 326-413 of human β2AR). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody. Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP-β2AR.

(C) Quantification of (B) (***p<0.001, ANOVA).

(D) Pull-Down of GST-CTC but not GST-CTD or GST-CTE (middle IB) by immobilized MBP-β2AR C-terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody.

(E) Quantification of (D) (***p<0.001, ANOVA).

(F) IP of β2AR in the presence of 10 μM GST (Control; Ctr) or GST-tagged C-terminal fragments as indicated. CT4 and CTC specifically displaced α11.2 (top of IB) but not GluA1 (middle of same IB) from β2AR (bottom of same IB). Use of non-specific IgG (left lane in right panels) indicates specificity of IP.

(G, H). For quantification of coIPs, α11.2 and GluA1 immunosignals were normalized to β2AR signals (***p<0.001, ANOVA).

(I) Representative IB showing amounts of the GST fusion proteins that were added in (F), as detected by anti-GST antibodies.
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 2

Figure 2. The β2AR Binds to the S1928 Phosphorylation Site.

(A) Titration of fluorescence polarization of FITC-peptides (100 nM) spanning α11.2 aa 1906-1959 with purified MBP-β2AR C-terminus. Kd value was obtained by fitting a non-linear direct binding curve to Pep2.

(B) IP of β2AR in the absence (Control) or presence of 10 μM peptides, as indicated. Pep2 specifically displaced α11.2 (top of IB) but not GluA1 (middle, same IB) from β2AR (bottom, same IB). Use of non-specific IgG (left lane) indicates specificity of (co)IPs.

(C, D). For quantification, α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).

(E) IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displaced α11.2.

(F) α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 3

Figure 3. S1928 Phosphorylation Displaces β2AR from Cav1.2.

Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).

(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).

(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).

(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).

(E,F,K,L) For quantification of coIP, α11.2 and GluA1 signals were normalized to β2AR (***p<0.001, One Way ANOVA).
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 4

Figure 4. S1928 Phosphorylation Separates β2AR from Cav1.2.

Hippocampal cultures were transfected with FLAG-β2AR and α11.2-HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 M isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.

(A,B) Representative immunofluorescent images obtained by wide-field microscopy at lower and higher resolutions (scale bar in A is 5 m).

(C) Quantification of distance between centers of HA and FLAG puncta (**p<0.001, Mann-Whitney Rank Sum Test).