Danaher Inc anti cardiac troponin i3

Morphological analysis of EHTs in four different media at day 21 of culture. ( A ) Brightfield images of full EHTs in BPEL+HS, DMEM+HS, MM+HS, or MM(SF). Scale bar = 0.5 mm. ( B ) Confocal images of whole-mount tissue immunostaining for cardiac <t>troponin</t> T (cTnT, red) counterstained with DAPI (nuclei, blue). Scale bars, 25 µm. HS, horse serum and SF, serum-free.

Anti Cardiac Troponin I3, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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jak (Danaher Inc)

Danaher Inc jak

Jak, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti bace1 (Abcam)

Abcam rabbit anti bace1

<t>BACE1</t> and Kv3.4 colocalize in the MF pathway of the hippocampus. A, Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B, Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C, Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B.

Rabbit Anti Bace1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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goat anti-horse igg h&l (Advisains)

Advisains goat anti-horse igg h&l

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Image Search Results

Journal: Journal of Personalized Medicine

Article Title: A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues

doi: 10.3390/jpm12020214

Figure Lengend Snippet: Morphological analysis of EHTs in four different media at day 21 of culture. ( A ) Brightfield images of full EHTs in BPEL+HS, DMEM+HS, MM+HS, or MM(SF). Scale bar = 0.5 mm. ( B ) Confocal images of whole-mount tissue immunostaining for cardiac troponin T (cTnT, red) counterstained with DAPI (nuclei, blue). Scale bars, 25 µm. HS, horse serum and SF, serum-free.

Article Snippet: Primary antibody anti-cardiac troponin T (1:400; Invitrogen, MA5-12960), anti-connexin-43 (1:200; Sigma-Aldrich, C6219), or anti-cardiac troponin I3 (1:800; Abcam, ab10231) were then incubated for 2 days at 4 °C.

Techniques: Immunostaining

BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A, Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B, Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C, Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B.

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A, Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B, Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C, Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B.

Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na + -K + -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R&D Systems), mouse-anti-K Ca 1.1 (ab192759; Abcam), rabbit-anti-K Ca 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; Bhattacharyya et al., 2013 ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

Techniques: Immunofluorescence, Staining, Double Staining

Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A, Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B, Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C, Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na+-K+-ATPase and KO samples were normalized to WT according to B. Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, *p < 0.05, **p < 0.01, two-tailed one-sample t test.

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A, Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B, Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C, Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na+-K+-ATPase and KO samples were normalized to WT according to B. Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, *p < 0.05, **p < 0.01, two-tailed one-sample t test.

Techniques: Western Blot, Positive Control, Two Tailed Test

BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A, Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B–D, Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B, This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C, D, Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. ***p < 0.001, two-tailed Mann–Whitney test applied to all unbinned vesicle velocities.

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A, Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B–D, Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B, This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C, D, Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. ***p < 0.001, two-tailed Mann–Whitney test applied to all unbinned vesicle velocities.

Techniques: Cell Culture, Immunofluorescence, Double Staining, Transfection, Two Tailed Test, MANN-WHITNEY

$BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A, Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B, Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K+ channels and synaptic markers. n = 3 for each genotype, *p < 0.05, **p < 0.01, two-tailed two-sample t test.$

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A, Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B, Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K+ channels and synaptic markers. n = 3 for each genotype, *p < 0.05, **p < 0.01, two-tailed two-sample t test.

Techniques: Western Blot, Two Tailed Test

$Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A, Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B, Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C, Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D, Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, **p < 0.01, ***p < 0.001. n.s., Not significant, two-tailed two-sample t test.$

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A, Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B, Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C, Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D, Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, **p < 0.01, ***p < 0.001. n.s., Not significant, two-tailed two-sample t test.

Techniques: Western Blot, Marker, Expressing, Two Tailed Test

Reduced Kv3 current leads to altered synaptic transmission in BACE1 KO. A, MFs from BACE1 KO mice manifested a reduced I/O relationship for MF-fEPSP. Peak amplitudes were plotted as a function of stimulus intensity. Corresponding fiber volley amplitudes are depicted in the inset. WT, n = 16 slices; KO, n = 18 slices. B, C, I/O curves determined after 2 min (control) and 30 min with 100 μm 4-AP (B) or 30 μm BDS-I (C) in the recording pipette and plotted as described above. The inserts in B depict MF-fEPSP traces from BACE1 WT and KO mice showing responses at 150 μA, 2 min after pipette insertion (black), and after 30 min (blue). WT, n = 7 slices; KO, n = 5 slices (B); WT, n = 5 slices; KO, n = 7 slices (C) D, Histograms summarizing the magnitude of drug effect on MF-fEPSP and MF-fiber volley at a stimulating intensity of 150 μA for WT and KO. E, Insert, Typical MF-fEPSP in response to 5 stimuli at 20 Hz (50 ms interspike interval) recorded from WT. The diagram shows the normalized MF-fEPSP amplitude relative to that of the first MF-fEPSP. WT, n = 15 slices; KO, n = 18 slices. F, Train stimuli determined after 5 min (control) and 35 min with 30 μm BDS-I in the recording pipette and plotted as in E. WT, n = 5 slices; KO, n = 7 slices. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed two-sample t test (A, D, E), two-tailed paired-sample t test (B, C, F).

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: Reduced Kv3 current leads to altered synaptic transmission in BACE1 KO. A, MFs from BACE1 KO mice manifested a reduced I/O relationship for MF-fEPSP. Peak amplitudes were plotted as a function of stimulus intensity. Corresponding fiber volley amplitudes are depicted in the inset. WT, n = 16 slices; KO, n = 18 slices. B, C, I/O curves determined after 2 min (control) and 30 min with 100 μm 4-AP (B) or 30 μm BDS-I (C) in the recording pipette and plotted as described above. The inserts in B depict MF-fEPSP traces from BACE1 WT and KO mice showing responses at 150 μA, 2 min after pipette insertion (black), and after 30 min (blue). WT, n = 7 slices; KO, n = 5 slices (B); WT, n = 5 slices; KO, n = 7 slices (C) D, Histograms summarizing the magnitude of drug effect on MF-fEPSP and MF-fiber volley at a stimulating intensity of 150 μA for WT and KO. E, Insert, Typical MF-fEPSP in response to 5 stimuli at 20 Hz (50 ms interspike interval) recorded from WT. The diagram shows the normalized MF-fEPSP amplitude relative to that of the first MF-fEPSP. WT, n = 15 slices; KO, n = 18 slices. F, Train stimuli determined after 5 min (control) and 35 min with 30 μm BDS-I in the recording pipette and plotted as in E. WT, n = 5 slices; KO, n = 7 slices. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed two-sample t test (A, D, E), two-tailed paired-sample t test (B, C, F).

Techniques: Transmission Assay, Transferring, Two Tailed Test

BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 (A1) or Kv3.4 with BACE1 (A2) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing (B1) or Kv3.4 + BACE1-expressing (B2) cell recorded with the inactivation protocol shown in the inset. C, The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D, Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV (B1, B2, prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2). Kv3.4, n = 49; + BACE1, n = 36. E, Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current (C). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol (B1) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F, Activation and inactivation time constants were estimated from recordings with the activation protocol (A1). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G, Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δt = 2i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation (IΔt) normalized to the peak before channel inactivation (I). Kv3.4, n = 44; + BACE1, n = 23. H, Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I, Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H. Kv3.4, n = 50; + BACE1, n = 42. D, E Inactivation, F, G, I, Cells with peak currents <2 nA at +80 mV in the activation protocol (A1) were excluded from analysis. Statistics: **p < 0.01, ***p < 0.001, normally distributed data were tested using a two-tailed two-sample t test (F); datasets showing no normal distribution were tested using a two-tailed Mann–Whitney test (C, D, G, I).

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 (A1) or Kv3.4 with BACE1 (A2) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing (B1) or Kv3.4 + BACE1-expressing (B2) cell recorded with the inactivation protocol shown in the inset. C, The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D, Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV (B1, B2, prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2). Kv3.4, n = 49; + BACE1, n = 36. E, Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current (C). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol (B1) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F, Activation and inactivation time constants were estimated from recordings with the activation protocol (A1). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G, Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δt = 2i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation (IΔt) normalized to the peak before channel inactivation (I). Kv3.4, n = 44; + BACE1, n = 23. H, Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I, Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H. Kv3.4, n = 50; + BACE1, n = 42. D, E Inactivation, F, G, I, Cells with peak currents <2 nA at +80 mV in the activation protocol (A1) were excluded from analysis. Statistics: **p < 0.01, ***p < 0.001, normally distributed data were tested using a two-tailed two-sample t test (F); datasets showing no normal distribution were tested using a two-tailed Mann–Whitney test (C, D, G, I).

Techniques: Expressing, Activation Assay, Generated, Transformation Assay, Two Tailed Test, MANN-WHITNEY

BACE1 interacts directly with Kv3.4. A, B, Representative Western blot of a Kv3.4-IP with BACE1 co-IP (A, n = 3) or BACE1-IP with Kv3.4 co-IP (B, n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C, FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t)= A * e−t/τ + I0 are shown. D, Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. *p < 0.05, **p < 0.01, one-way ANOVA with post hoc Tukey test. For illustration, time constant means ± SEM were back-transformed to a linear scale. + mCherry, n = 46; + Kv3.4, n = 29; + ENaC-mCherry, n = 31.

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: BACE1 interacts directly with Kv3.4. A, B, Representative Western blot of a Kv3.4-IP with BACE1 co-IP (A, n = 3) or BACE1-IP with Kv3.4 co-IP (B, n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C, FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t)= A * e−t/τ + I0 are shown. D, Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. *p < 0.05, **p < 0.01, one-way ANOVA with post hoc Tukey test. For illustration, time constant means ± SEM were back-transformed to a linear scale. + mCherry, n = 46; + Kv3.4, n = 29; + ENaC-mCherry, n = 31.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Marker, Fluorescence, Construct, Transformation Assay

BACE1 increases Kv3.4 surface level independent of its proteolytic activity in HEK293T cells. A, Representative Western blot of a surface biotinylation experiment of HEK293T cells. Transfected constructs and treatment with 5 μm BACE1 inhibitor IV are depicted above the blot. EGFP served as transfection marker. B, Total levels of Kv3.4 normalized to the corresponding total levels of β-actin, with the normalized levels determined in the absence of BACE1 set to 1. C, Surface levels of Kv3.4 normalized to the corresponding surface levels of pan-cadherin, with the normalized levels determined in the absence of BACE1 set to 1. n = 5, *p < 0.05, two-tailed one-sample t test with Bonferroni correction.

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

doi: 10.1523/JNEUROSCI.2643-17.2018

Figure Lengend Snippet: BACE1 increases Kv3.4 surface level independent of its proteolytic activity in HEK293T cells. A, Representative Western blot of a surface biotinylation experiment of HEK293T cells. Transfected constructs and treatment with 5 μm BACE1 inhibitor IV are depicted above the blot. EGFP served as transfection marker. B, Total levels of Kv3.4 normalized to the corresponding total levels of β-actin, with the normalized levels determined in the absence of BACE1 set to 1. C, Surface levels of Kv3.4 normalized to the corresponding surface levels of pan-cadherin, with the normalized levels determined in the absence of BACE1 set to 1. n = 5, *p < 0.05, two-tailed one-sample t test with Bonferroni correction.

Techniques: Activity Assay, Western Blot, Transfection, Construct, Marker, Two Tailed Test

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