Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Binding between AKAP79/AKAP150 and the N terminus of AC8. A , GST pull-downs using whole cell lysate from cells transiently expressing tagged AKAP proteins. Lane 1 , GST alone; lanes 2–4 , the following regions of AC8 fused to GST: 1–179 (N terminus), 582–703 (C1b domain), and 1106–1248 (C terminus), respectively. Lane 5 , input control (5%) to confirm expression of each construct. B , top , AKAP79-HA and AKAP150-HA transiently expressed in HEK293 cells were pulled down using GST-fused first half (residues 1–77) or second half (residues 73–179) of the AC8 N terminus. Bottom , immunoblotting of the GST-fused proteins used in the pull-downs in the top . The upper GST bands represent the full-length form of each protein. C , plot of densitometries from 3–6 repeats of blots presented in B to quantify binding of AKAP79/150 to GST-AC8 1–77 versus GST-AC8 73–179. Data are normalized to the intensity of the uppermost GST bands. Error bars , S.E. D , schematic diagram of GST-AC8 constructs used in A–C .

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Binding Assay, Expressing, Control, Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: AKAP disruption enhances CCE-induced AC8 activity. A , CCE-mediated increases in cAMP assessed using the FRET-based sensor Epac2-camps following knockdown of endogenous AKAP79 levels. Data are plotted as relative FRET ratio changes normalized to maximum signal response. Cells were pretreated with 200 n m Tg in Ca 2+ -free conditions, and CCE was induced upon the addition of 2 m m Ca 2+ to the bath solution. B , average Fura-2 traces from HEK-AC8 cells during CCE following transfection with AKAP150-HA, AKAP79-HA, or shRNA AKAP79. All data are normalized to maximal Fura-2 signal obtained upon the addition of 5 μ m ionomycin (Ca 2+ ionophore) and 5 m m Ca 2+ . C , effects of the AKAP/PKA disruptor peptide, St-Ht31 (10 μ m ), on CCE-stimulated AC8 activity assessed using Epac2-camps. Experiments were performed in the presence of 100 μ m IBMX, and CCE was induced upon the addition of 0.5 m m Ca 2+ . St-Ht31P (10 μ m ) was used as a negative control. D , analyses of the effects of AKAP79 knockdown or pharmacological AKAP disruption (St-Ht31) on AC8 activity. Plots of peak CCE-induced cAMP increase relative to control ( left chart ) and peak rate of cAMP production ( right chart ) are presented as mean ± S.E. ( error bars ). **, p < 0.001 using Students' t test.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Disruption, Activity Assay, Knockdown, Transfection, shRNA, Negative Control, Control

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Co-immunoprecipitation of endogenously expressed AC8 and AKAP150 in MIN6 cells. A , Western blot analysis to confirm the endogenous expression of AKAP150 ( left ) and AC8 ( right ) in MIN6 cells. B , immune complexes for endogenous AKAP150 co-purify with AC8 in MIN6 cell lysate. No AC8 band is seen in IgG controls. Antibodies used for immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) were specific to AKAP150 and AC8, respectively.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Effects of AKAP150 on Ca 2+ -stimulated AC8 activity in MIN6 cells. A , Fura-2 data showing the standard protocol for inducing CCE in MIN6 cells. Cells were pretreated with 1 μ m Tg in Ca 2+ -free conditions for 3 min prior to the addition of 2 m m external Ca 2+ . The addition of 100 μ m 2-aminoethoxydiphenyl borate ( 2-APB ; a CCE inhibitor when used at high concentrations) from 1 min onward significantly reduced Ca 2+ entry (see bar chart inset ; ***, p < 0.001). Bi , Epac2-camps data showing the effects of AKAP150 overexpression on Ca 2+ -stimulated cAMP production compared with empty vector controls. 20 n m FSK and 100 μ m IBMX were present throughout. Data are plotted as a percentage of maximal FRET signal obtained using saturating cAMP concentrations. Bii , as above, except that the effects of lentiviral shRNA directed against AKAP150 were compared with scrambled shRNA controls. C , bar charts show mean ± S.E. values ( error bars ) for peak amplitude and rate of FRET ratio changes during CCE for data in B . *, p < 0.05 compared with shRNA controls.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Over Expression, Plasmid Preparation, shRNA

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: A role for AKAP150 in the regulation of Ca 2+ -stimulated AC activity in hippocampal neurons. A , the basic design of the citrine-Epac2-camps-CFP (Ci/C-Epac2-camps) sensor used for hippocampal experiments and fluorescent images taken from three individual hippocampal neurons showing cytosolic expression of the cAMP sensor. Scale bar , 20 μm. B , in vitro calibrations of the Ci/C-Epac2-camps compared with the original Epac2-camps. Note the reduced pH sensitivity of the citrine-CFP version. C , comparison of Ca 2+ -stimulated AC activity in control, AKAP150-HA-expressing, and AKAP150 knockdown hippocampal neurons assessed using Ci/C-Epac2-camps. 1 μ m FSK was added in Ca 2+ -free conditions at 120 s with the readdition of 2 m m external Ca 2+ at 180 s to monitor Ca 2+ -dependent cAMP production. Maximum FRET ratio change was obtained by the subsequent addition of 10 μ m FSK, 10 μ m isoproterenol, and 100 μ m IBMX. D , overlay of control, AKAP150 overexpression, and shRNA AKAP150 data to compare Ca 2+ -stimulated AC activities. E , data analysis showing significant delay in Ca 2+ stimulation of cAMP production in neurons overexpressing AKAP150. Data represent mean ± S.E. ( error bars ) for each condition. n values are indicated on the graph . *, p < 0.01.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Expressing, In Vitro, Comparison, Control, Knockdown, Over Expression, shRNA

Journal: Protein Science : A Publication of the Protein Society

Article Title: Rapid directed molecular evolution of fluorescent proteins in mammalian cells

doi: 10.1002/pro.4261

Figure Lengend Snippet: Intracellular brightness and photostability of mCardinal, TagRFP658, and emiRFP2 in live cultured hippocampal mouse neurons and in vivo in zebrafish larvae. (a,b) Representative fluorescence images of primary cultured mouse hippocampal neurons expressing TagRFP658 at (a) 14 and (b) 23 days in vitro (DIV; n = 53 and 33 neurons, respectively, from two independent cultures). Imaging condition: excitation 631/28 nm from an LED, emission 664LP. (c) Representative light sheet image of head of zebrafish larvae at 4 days postfertilization expressing TagRFP658 in neurons ( n = 10 fish from two independent injections). Imaging conditions: excitation 638 nm from a laser, emission 665LP. (i, ii) High‐magnification images of the respective regions shown in white boxes in e. Scale bars, 50 μm. (d) Relative fluorescence of cultured mouse hippocampal neurons expressing mCardinal and TagRFP658 ( n = 78 and 85 neurons, respectively, from two independent cultures for each protein; one‐way analysis of variance [ANOVA]). Imaging conditions as in (a). Box plots with notches are used in this figure (see Figure for the full description). (e) Raw photobleaching curves for mCardinal (dashed line) and TagRFP658 (solid line) in primary cultured mouse hippocampal neurons ( n = 9 and 7 neurons, respectively, from one culture each; one‐way ANOVA). Imaging condition: excitation 631/28 nm from an LED at 70 mW/mm 2 , emission 664LP. (f) Representative fluorescence images of cells transfected with pAAV‐CAG‐mCardinal‐P2A‐GFP (top), pAAV‐TagRFP658‐P2A‐GFP (middle), and pAAV‐emiRFP2‐P2A‐GFP ( n = 39, 33, and 41 neurons from three, two, and three independent transfections from one culture each for mCardinal, TagRFP658, and emiRFP2, respectively, for Cy5 channel and n = 15 and neurons from one independent transfection from one culture each for mCardinal and emiRFP2, respectively, for Cy5.5 channel). Imaging conditions: Cy5 channel: excitation 635/22 nm from 637 nm laser, emission 730/140 nm; Cy5.5 channel: excitation 680/13 nm from 680 nm laser, emission 710 LP; GFP channel: excitation 478/24 nm for an LED; emission 535/46 nm. Images in Cy5 and Cy5.5 were taken under matching excitation intensity (66 mW/mm 2 ) and the same exposure time (100 ms). The dynamic range of fluorescence intensity in Cy5 and Cy5.5 channels are identical across all images. Scale bar, 20 μm. (g) Near‐infrared (NIR)‐to‐green fluorescence ratio for mCardinal, TagRFP658, and emiRFP2 for the experiment shown in (f). (h) Intracellular photostability of mCardinal, TagRFP658, and emiRFP2 in Cy5 and Cy5.5 channels ( n = 8, 7, and 9 neurons from three, two, and three independent transfections from one culture each for mCardinal, TagRFP658, and emiRFP2, respectively, under Cy5 excitation and n = 5 neurons from one transfection from one culture for emiRFP2 under Cy5.5 excitation). Imaging conditions the same as in (a)

Article Snippet: Mouse primary hippocampal neurons were prepared from postnatal day 0 or 1 Swiss Webster (Taconic) mice (both male and female mice were used) and cultured as previously described.

Techniques: Cell Culture, In Vivo, Fluorescence, Expressing, In Vitro, Imaging, Transfection

Journal: ACS chemical neuroscience

Article Title: Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability

doi: 10.1021/acschemneuro.8b00360

Figure Lengend Snippet: Assessment of several terminal protein expression levels. Relative protein expression levels of (A) VMAT2 (n = 7 in both groups), (B) Synaptigyrin-3 (aGH: n = 6; aSI: n = 5), (C) Syntaxin-1 (aGH: n = 7; aSI: n = 6), and (D) Munc13-3 (aGH: n = 8; aSI: n = 8) were measured. None of these proteins had different expression levels in aGH and aSI rats. (insets) The representative Western blot images, with the respective protein and actin for comparison. Group housed, aGH, blue; Socially isolated, aSI, red.

Article Snippet: Subsequently, blots were incubated with agitation for 2 h at room temperature in TBS-T/5% bovine serum albumin (05470; Sigma-Aldrich) solution containing the following primary antibody concentrations: VAMT2 (1:2000; AB1598P; Millipore Sigma); Synaptogyrin-3 (1:1000; ab106460; abcam); Syntaxin-1 (1:1000; ANR-002; Alomone laboratories).

Techniques: Expressing, Western Blot, Isolation

Journal: International journal of molecular sciences

Article Title: Role of Luteolin as Potential New Therapeutic Option for Patients with Glioblastoma through Regulation of Sphingolipid Rheostat.

doi: 10.3390/ijms25010130

Figure Lengend Snippet: Figure 4. Effect of increasing doses of luteolin on astrocytes (A), NPC (B) and NPSC (C). No significant decreases in cell viability were observed, not even at the highest dose tested. Data are the mean ± standard deviation of at least three experiments, run in triplicate.

Article Snippet: Astrocytes were purchased by ABM (Cat. N. T0280), while NPC and NPSC were purchased by CelProgen (Cat. N.: 36057-01 and 36057-02).

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Behavioral state regulates the dynamics of memory consolidation

doi: 10.1101/2024.06.28.601231

Figure Lengend Snippet: (A) Diagram of cerebellar cortex (green), targeted here for reversible inactivations. (B) Schematic of guide cannula (grey) chronically implanted at cerebellar surface and internal cannula (white) acutely introduced after each training session to deliver muscimol in the eyelid area of cerebellar cortex. (C) Coronal brain slice. Purkinje cells stained for calbindin (red); fluorescent muscimol (GABAa receptor agonist), green. (D) Protocol for post-session reversible inactivations. Muscimol or vehicle (control) was infused after each session. Learning was assessed when training resumed 24h later with the subsequent session. (E) Left: Averaged acquisition curves (± s.e.m.) of mice trained on a self-paced running wheel, treated with vehicle-(black, N=10) or muscimol (green, N=15) after each training session. p =0.0002***; repeated-measures-ANOVA. Center: After training completion, muscimol was infused before test sessions (M), to verify cannula placement; Controls (black): p=0.00015***; Muscimol (green): p=0.0008***; paired-t-tests, before vs. after muscimol. Performance was not impaired with vehicle infusion (V) in a subsequent session; Controls (black): p=0.5n.s; Muscimol (green): p=0.2n.s.; paired-t-tests, before vs. after vehicle. Right: Controls then received muscimol infusions after additional training sessions to test the effect of reversible inactivations after acquisition (plateau sessions, N=5). (F) Quantification of learning onset session (see Methods). p=0.02*; Mann-Whitney-Wilcoxon test. Non-learners (NL) did not exhibit CRs within 14 training sessions. Each dot is one mouse. (G) Averaged eyelid closures (± s.e.m.) for CS-only trials from the last training sessions. Amplitudes: p=0.046*, controls (black, N=10) vs. muscimol (green, N=15), t-test. Blue: visual CS. Red dashed line and shading: timing of the (omitted) airpuff-US. (H) Same as (E) but for mice trained while locomoting at a constant speed (0.1 m/s) on a motorized running wheel (Vehicle controls, black, N=14; Muscimol, green, N=12). Left: Acquisition, p=0.7n.s.; repeated-measures-ANOVA. Middle: Before vs. after Muscimol test (M). Controls: p=1.7e-6***, Muscimol, p=2.9e-6***; paired-t-tests. Right: Before vs. after Vehicle test (V). Controls, p=0.4n.s; Muscimol, p=0.1n.s., paired t-tests. (I) Same as (F) but for mice in (H); Controls vs. Muscimol, p=0.5n.s., Mann-Whitney-Wilcoxon test. (J) Same as (G) but for mice in (H); Controls vs. Muscimol, p=0.9n.s., t-test.

Article Snippet: Brain sections (50um thick) were cut in a vibratome and stained for Purkinje cells (with chicken anti-calbindin primary antibody #214006 SYSY, and anti-chicken Alexa488 #703-545-155 or Alexa594 #703-545-155 secondary antibodies from Jackson Immunoresearch) and for cell viability (with DAPI marker).

Techniques: Slice Preparation, Staining, Control, MANN-WHITNEY