ip3r2  (Alomone Labs)

Journal: bioRxiv

Article Title: Astrocyte Store-Released Calcium Modulates Visual Cortex Synapse Development and Circuit Function

doi: 10.1101/2025.07.20.665758

Figure Lengend Snippet: See also Fig. S6. A. Schematic of the strategy to express fluorescent reporter in astrocytes. P1 mouse pups were injected icv with AAV to express membrane tethered eGFP (Lck-eGFP) under astrocyte specific promoter GfaABC1D. Tissue is collected 2 weeks following injection, sectioned and imaged using Airyscan super resolution confocal microscope. B-E. Astrocytic volume is reduced in IP3R2 KO mice compared to WT. Example images of Lck-eGFP (green) expressing astrocytes ( D ) and 3D rendering of volume using Imaris ( E ) for each genotype is shown as labeled. B-C. Quantification shows reduced total volume and area of IP3R2 KO astrocytes in L1 VC. Graphs show mean ± s.e.m. Black circles above each bar are average of signal in each mouse, colored open circles are data for each astrocyte. Number of mice/ group (N) N=5, number of astrocytes = 25-27. Scale bar = 10 μm. **P<0.01 by t-test.

Article Snippet: The following primary antibodies were used: Rb anti IP3R2 (Alomone labs #ACC-116, 1:250), Gp anti-VGLUT1 (Millipore #AB5905, 1:1000), Gp anti-VGLUT2 (Millipore #AB2251 1:1000), Rb anti-PSD95 (Fisher #516900 1:250), Gp anti-VGAT (Synaptic Systems #131004 1:250), Rb anti-Gephyrin (Synaptic Systems #147008 1:500), Rb anti-Nf200 (Millipore Sigma #N4142 1:400), Chk anti-GFP (Invitrogen A10262 1:1000), Rb anti-S100β (Abcam #AB52642, 1:100).

Techniques: Injection, Membrane, Microscopy, Expressing, Labeling

Journal: bioRxiv

Article Title: Astrocyte Store-Released Calcium Modulates Visual Cortex Synapse Development and Circuit Function

doi: 10.1101/2025.07.20.665758

Figure Lengend Snippet: See also Fig S1. A. Schematic of experiment: Brain tissue is collected from WT and KO mice at P7, P14 and P28 corresponding to stages of synapse development; IHC to quantify synapses as indicated is performed in Layer 1 of the VC for VGLUT1-containing cortico-cortical synapses, and VGLUT2-containing thalamo-cortical synapses. B. Validation of IP3R2 KO by IHC and WB (top right panel). Example images of IP3R2 (cyan), astrocyte marker S100ꞵ (magenta) and neuronal marker Neun (blue) in the VC at P14 as labeled. Graph on the right is quantification of colocalized signal with each cell marker. IP3R2 signal is highly colocalized with astrocytes and not with neurons and is downregulated in KO VC. WB shows IP3R2 band (∼250KDa) and GAPDH (loading control, ∼36 KDa) in WT and KO at P14 as labeled. Numbers indicate samples from individual animals. See also Fig. S1A-D. C-G. Cortico-cortical VGLUT1-containing, synapses are reduced in IP3R2 KO VC at P14 and P28 but not P7. Example images of the presynaptic VGLUT1, postsynaptic PSD95 and merged (synapses) in each age and genotype as labeled ( C-D ) and quantification of individual synaptic proteins and synapse number per mm 3 represented as colocalization between VGLUT1 and PSD95 for both genotypes ( E-F ) are shown. Single channel grayscale images on the left, merged images on the right. The number of VGLUT1 puncta and VGLUT1-containing synapses is reduced in KO ( E, G ), while PSD95 numbers are unaltered ( F ). H-J. Thalamo-cortical VGLUT2-containing synapses are reduced in IP3R2 KO VC at P14 and P28 but not P7. Quantification of individual synaptic proteins and synapse number per mm 3 represented as colocalization between VGLUT2 and PSD95 for both genotypes are shown (see also Fig. S1E-F). The number of VGLUT2 puncta and VGLUT2-containing synapses is reduced in KO ( H, J ), while PSD95 numbers are unaltered ( I ). Plots show mean ± s.e.m. Squares and circles above each bar are average of signal in each mouse. Number of mice/ group (N) N=5. Scale bar = 5 μm. Arrowheads mark representative colocalized puncta. #,*P<0.05, ##, **P<0.01, ###, ***P<0.001. # indicates comparing age groups within each genotype by one-way ANOVA. Within each age, WT and KO comparison by t-test indicated by *. ns denotes Non-significant results (P>0.05).

Article Snippet: The following primary antibodies were used: Rb anti IP3R2 (Alomone labs #ACC-116, 1:250), Gp anti-VGLUT1 (Millipore #AB5905, 1:1000), Gp anti-VGLUT2 (Millipore #AB2251 1:1000), Rb anti-PSD95 (Fisher #516900 1:250), Gp anti-VGAT (Synaptic Systems #131004 1:250), Rb anti-Gephyrin (Synaptic Systems #147008 1:500), Rb anti-Nf200 (Millipore Sigma #N4142 1:400), Chk anti-GFP (Invitrogen A10262 1:1000), Rb anti-S100β (Abcam #AB52642, 1:100).

Techniques: Biomarker Discovery, Marker, Labeling, Control, Comparison

Journal: bioRxiv

Article Title: Astrocyte Store-Released Calcium Modulates Visual Cortex Synapse Development and Circuit Function

doi: 10.1101/2025.07.20.665758

Figure Lengend Snippet: See also Fig. S4. A. Schematic of experimental paradigm. Following 4 hours of dark exposure (ZT12 marks lights off), mice were exposed to 20-minute light pulse, tissue collected immediately after for IHC analysis of c-FOS expression. B, E. Light evoked c-FOS levels are diminished in IP3R2 KO mice VC. Example images of c-FOS (green) and nuclear marker DAPI (blue) in each genotype as labeled in the dark or light exposed groups. Neuronal cortical layers are labeled on the right E. Quantification of B represented as c-FOS positive cell numbers per area. Light exposure produced a strong increase in the number of c-FOS positive cells in the WT, but not in the KO. C-G. Same as B, E, but for the dorsal lateral geniculate nucleus of the thalamus (dLGN; C, F) and the superior colliculus (SC; D, G). In both regions, light exposure produced a strong increase in the number of c-FOS positive cells in the WT, but to a lesser extent in the KO. Graphs show mean ± s.e.m. Squares and circles above each bar are average of signal in each mouse. Number of mice/ group (N) N=5. Scale bar in B = 100 μm; in C-D = 20 μm. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA. ns denotes non-significant results (P>0.05).

Article Snippet: The following primary antibodies were used: Rb anti IP3R2 (Alomone labs #ACC-116, 1:250), Gp anti-VGLUT1 (Millipore #AB5905, 1:1000), Gp anti-VGLUT2 (Millipore #AB2251 1:1000), Rb anti-PSD95 (Fisher #516900 1:250), Gp anti-VGAT (Synaptic Systems #131004 1:250), Rb anti-Gephyrin (Synaptic Systems #147008 1:500), Rb anti-Nf200 (Millipore Sigma #N4142 1:400), Chk anti-GFP (Invitrogen A10262 1:1000), Rb anti-S100β (Abcam #AB52642, 1:100).

Techniques: Expressing, Marker, Labeling, Produced

Journal: bioRxiv

Article Title: Astrocyte Store-Released Calcium Modulates Visual Cortex Synapse Development and Circuit Function

doi: 10.1101/2025.07.20.665758

Figure Lengend Snippet: A. Diagram depicting inhibitory neurons within the VC analyzed. B-F. GABAergic synapse numbers are not altered in IP3R2 KO VC at P14. Example images of the presynaptic VGAT, postsynaptic Gephyrin and merged (synapses) in each genotype as labeled ( B ) and quantification of individual synaptic proteins and synapse number per mm 3 represented as colocalization between VGAT and Gephyrin for both genotypes ( C-E ) are shown. Single channel grayscale images on the left, merged images on the right. No difference is observed in any of the parameters compared. F. Cumulative distributions of volumes from 3D rendered images for VGAT per genotype as labeled. Bar graphs show mean ± s.e.m. Squares and circles above each bar are average of signal in each mouse. Number of mice/ group (N) N=5. Scale bar = 5 μm. Arrowheads mark representative colocalized puncta. ns denotes non-significant results (P>0.05) by t-test in C-E, and Kolmogorov-Smirnov test in F, comparing WT and KO groups.

Article Snippet: The following primary antibodies were used: Rb anti IP3R2 (Alomone labs #ACC-116, 1:250), Gp anti-VGLUT1 (Millipore #AB5905, 1:1000), Gp anti-VGLUT2 (Millipore #AB2251 1:1000), Rb anti-PSD95 (Fisher #516900 1:250), Gp anti-VGAT (Synaptic Systems #131004 1:250), Rb anti-Gephyrin (Synaptic Systems #147008 1:500), Rb anti-Nf200 (Millipore Sigma #N4142 1:400), Chk anti-GFP (Invitrogen A10262 1:1000), Rb anti-S100β (Abcam #AB52642, 1:100).

Techniques: Labeling

Journal: Science Advances

Article Title: A pleiotropic recurrent dominant ITPR3 variant causes a complex multisystemic disease

doi: 10.1126/sciadv.ado5545

Figure Lengend Snippet: ( A ) ITPR3 mRNA expression in patient 1’s HDFs ( n = 2 independent experiments), patient 2’s PBMCs ( n = 3), patient 4’s HDFs ( n = 3), and blood ( n = 3) versus those from various controls and ( B ) in CRISPR-Cas9–edited Jurkat cells ( n = 3, KI = IP3R3 R2524C knock-in, KO = IP3R3 knock-out) as measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR), normalized to housekeeping genes GAPDH and β -actin . ( C and D ) Representative Western blot of IP3R3 (top) and calnexin (bottom) in (C) patient 1’s HDFs versus age-matched control HDFs and (D) in five CRISPR-Cas9–edited Jurkat cells. Right [(C) and (D)]: Relative IP3R3 expression quantification by Western blot band intensity normalized to calnexin ( n = 3 independent experiments each). ( E and F ) Representative Western blot of IP3R2 (top) and calnexin (bottom) in (E) patient 1’s HDFs compared to control HDFs and (F) in the above-described five CRISPR-Cas9–edited Jurkat cells as well as in a negative control (IP3R2 CRISPR-Cas9 KO in Jurkat cells). Right [(E) and (F)]: Relative IP3R2 expression quantification by Western blot band intensity normalized to calnexin [ n = 3 for both (E) and (F)]. All values are represented as mean ± SD. Statistics: (A), (C), and (E): T test; (B), (D), and (F): One-way analysis of variance (ANOVA) [* P < 0.05, ** P < 0.01, *** P < 0.001, **** P <0.0001, and P > 0.05 (not shown)]. Data values are provided in Table S3.

Article Snippet: Cells were incubated for 1 hour and 30 min with either anti-IP3R3 antibody (mouse IgG; 610312, BD Biosciences; used at 1:500 dilution, Franklin Lakes, NJ) or anti-IP3R2 monoclonal antibody (mouse IgG; SC398434, Santa Cruz Biotechnology, Dallas, TX; used at 1:250 dilution) in PBS-Ca-Mg, 2% BSA, and, in some conditions, with anti-calnexin polyclonal antibody (rabbit IgG; PA5-34754, Thermo Fisher Scientific, Waltham, MA; used at 1:200 dilution) in PBS-Ca 2+ -Mg 2+ and 2% BSA.

Techniques: Expressing, CRISPR, Knock-In, Knock-Out, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control, Negative Control

Journal: Science Advances

Article Title: A pleiotropic recurrent dominant ITPR3 variant causes a complex multisystemic disease

doi: 10.1126/sciadv.ado5545

Figure Lengend Snippet: ( A ) Confocal microscopy images of patient 1 and age-matched control HDFs labeled against IP3R3 (left two columns) (green), IP3R2 (right two columns) (green), ER (top, magenta), or mitochondria (bottom, magenta). Each condition is represented as a whole cell image and a ×40 magnification of regions highlighted by yellow squares. All conditions were also labeled for nuclei (blue). Colocalizing regions appear in white. ( B ) Mitochondrial area normalized to the total cell area of patient 1 and age-matched control HDFs ( n = 50 and n = 64, respectively). ( C ) IP3R3 area normalized to the total cell area in patient 1 and age-matched control HDFs ( n = 59 and n = 59, respectively). ( D ) IP3R3 and ER area overlaps represented as total overlap count (left) and as percentage of whole cell IP3R3 area (right) (patient n = 31 and control n = 30). ( E ) IP3R3 and mitochondrial overlaps represented as the total overlap count (left) and as percentage of whole cell IP3R3 area (right) (patient n = 28 and control n = 29). ( F ) IP3R2 area normalized to the total cell area in patient 1 and age-matched control HDFs ( n = 60 and n = 75, respectively). ( G ) IP3R2 and ER overlaps represented as the total overlap count (left) and as the percentage of whole-cell IP3R2 area (right) (patient n = 37 and control n = 37). ( H ) IP3R2 and mitochondria overlaps represented as the total overlap count (left) and as a percentage of whole cell IP3R2 area (right) (patient n = 23 and control n = 38). All experiments were performed in triplicate. Mann-Whitney test was used. * P < 0.05, ** P < 0.01, and **** P < 0.0001. ns, not significant.

Article Snippet: Cells were incubated for 1 hour and 30 min with either anti-IP3R3 antibody (mouse IgG; 610312, BD Biosciences; used at 1:500 dilution, Franklin Lakes, NJ) or anti-IP3R2 monoclonal antibody (mouse IgG; SC398434, Santa Cruz Biotechnology, Dallas, TX; used at 1:250 dilution) in PBS-Ca-Mg, 2% BSA, and, in some conditions, with anti-calnexin polyclonal antibody (rabbit IgG; PA5-34754, Thermo Fisher Scientific, Waltham, MA; used at 1:200 dilution) in PBS-Ca 2+ -Mg 2+ and 2% BSA.

Techniques: Confocal Microscopy, Control, Labeling, MANN-WHITNEY