Journal: Respiratory Research

Article Title: Abnormal spatial diffusion of Ca 2+ in F508del-CFTR airway epithelial cells

doi: 10.1186/1465-9921-9-70

Figure Lengend Snippet: Specific primers for each IP 3 Rs subtype

Article Snippet: We used the following primary specific antibody for each IP 3 R isoform: rabbit anti-IP 3 R1 polyclonal antibody (1:1000, Affinity Bioreagents), goat anti-IP 3 R2 polyclonal antibody (1:1000, Santa Cruz Biotechnology), mouse anti-IP 3 R3 monoclonal antibody (1:1000, Santa Cruz Biotechnology) and the rabbit anti-calreticulin antibody (1:100, Stressgen Biotechnologies) for 1 h at room temperature.

Techniques:

Journal: Respiratory Research

Article Title: Abnormal spatial diffusion of Ca 2+ in F508del-CFTR airway epithelial cells

doi: 10.1186/1465-9921-9-70

Figure Lengend Snippet: Characterization of IP 3 Rs isoforms in human nasal epithelial cells . A mRNA amplification of 3 isoforms of IP 3 R by real time PCR. B Immunostaining of IP 3 R type 1, 2 and 3 in untreated CF15 cells and staining with the secondary antibody as a negative control (bottom panels); nuclei are labelled with TOPRO-3, bar = 10 μm.

Article Snippet: We used the following primary specific antibody for each IP 3 R isoform: rabbit anti-IP 3 R1 polyclonal antibody (1:1000, Affinity Bioreagents), goat anti-IP 3 R2 polyclonal antibody (1:1000, Santa Cruz Biotechnology), mouse anti-IP 3 R3 monoclonal antibody (1:1000, Santa Cruz Biotechnology) and the rabbit anti-calreticulin antibody (1:100, Stressgen Biotechnologies) for 1 h at room temperature.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Immunostaining, Staining, Negative Control

Journal: Respiratory Research

Article Title: Abnormal spatial diffusion of Ca 2+ in F508del-CFTR airway epithelial cells

doi: 10.1186/1465-9921-9-70

Figure Lengend Snippet: Pharmacology of IP 3 R response of local uncaging of caged Ca 2+ in CF15 cells in absence of extracellular Ca 2+ . A Example of line-scan images acquired at 2 ms per line and 0.21 μm per pixel in CF15 cells untreated at 37°C in presence or not of 100 μM 2-APB, 100 μM decavanadate, 20 mM caffeine or 10 μM cyclosporine A (all were preincubated during 10 min) and after 2 h incubation with 10 μM thapsigargin (TG). B Average of the line-scan images in A expressed as normalized fluorescence in each conditions C Mean normalized area measured from XT images in each experimental condition. The dash line represents the response induced by the flash only, after complete ER Ca 2+ store depletion. Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. * P < 0.05; ** P < 0.01*** P < 0.001; ns, non significant difference.

Article Snippet: We used the following primary specific antibody for each IP 3 R isoform: rabbit anti-IP 3 R1 polyclonal antibody (1:1000, Affinity Bioreagents), goat anti-IP 3 R2 polyclonal antibody (1:1000, Santa Cruz Biotechnology), mouse anti-IP 3 R3 monoclonal antibody (1:1000, Santa Cruz Biotechnology) and the rabbit anti-calreticulin antibody (1:100, Stressgen Biotechnologies) for 1 h at room temperature.

Techniques: Incubation, Fluorescence

Journal: Respiratory Research

Article Title: Abnormal spatial diffusion of Ca 2+ in F508del-CFTR airway epithelial cells

doi: 10.1186/1465-9921-9-70

Figure Lengend Snippet: Modification of local stimulation of caged Ca 2+ in corrected F508del-CFTR CF15 cells . A Relative mRNA expression level of IP 3 R-1, IP 3 R-2, and IP 3 R-3 in different conditions compared to βActin mRNA expression. B Example of line-scan images acquired at 2 ms per line and 0.21 μm per pixel in CF15 cells treated (27°C, miglustat, NB-DGJ and uncorrected at 37°C in absence of extracellular Ca 2+ ). C Average of the line-scan images in B expressed as normalized fluorescence in absence of extracellular Ca 2+ . D Histograms showing the amplitude of IP 3 Rs Ca 2+ response in various experimental conditions as indicated. E Mean normalized area in each experimental treatment in absence or presence of 10 μM CsA. Sets of data were compared to the control CF15. Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. ** P < 0.01, *** P < 0.001; ns, non significant difference.

Article Snippet: We used the following primary specific antibody for each IP 3 R isoform: rabbit anti-IP 3 R1 polyclonal antibody (1:1000, Affinity Bioreagents), goat anti-IP 3 R2 polyclonal antibody (1:1000, Santa Cruz Biotechnology), mouse anti-IP 3 R3 monoclonal antibody (1:1000, Santa Cruz Biotechnology) and the rabbit anti-calreticulin antibody (1:100, Stressgen Biotechnologies) for 1 h at room temperature.

Techniques: Modification, Expressing, Fluorescence

Journal: Respiratory Research

Article Title: Abnormal spatial diffusion of Ca 2+ in F508del-CFTR airway epithelial cells

doi: 10.1186/1465-9921-9-70

Figure Lengend Snippet: F508del-CFTR correction in CF-KM4 cells restored histamine ER Ca 2+ release compared to non CF MM39 cells . A mRNA amplification of 3 isoforms of IP 3 R by real time PCR in MM39 and CF-KM4 cells. B Example of line-scan images acquired in MM39 cells and in uncorrected or corrected CF-KM4 cells in absence of extracellular Ca 2+ . These cells were incubated 2 h at 37°C with 100 μM miglustat or 100 μM NB-DGJ. C Average of the line-scan images in A expressed as normalized fluorescence in each conditions. D Histogram of the normalized area under curve of intensity profile of Ca 2+ response extracted from A in various experimental conditions as indicated. E Mean of amplitude of Ca 2+ response in each experimental condition. Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. *** P < 0.001; ns, non significant difference.

Article Snippet: We used the following primary specific antibody for each IP 3 R isoform: rabbit anti-IP 3 R1 polyclonal antibody (1:1000, Affinity Bioreagents), goat anti-IP 3 R2 polyclonal antibody (1:1000, Santa Cruz Biotechnology), mouse anti-IP 3 R3 monoclonal antibody (1:1000, Santa Cruz Biotechnology) and the rabbit anti-calreticulin antibody (1:100, Stressgen Biotechnologies) for 1 h at room temperature.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Incubation, Fluorescence

Journal: Frontiers in Aging Neuroscience

Article Title: N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer’s Disease Rat Model

doi: 10.3389/fnagi.2018.00399

Figure Lengend Snippet: Hippocampal-derived MAM fractions contain RyR2, which is enriched in the MAM fraction from AβOs-injected rats. Western blot images of MAM fractions isolated from rat hippocampus (see Materials and Methods). The ER proteins analyzed were Calnexin (CNX), IP 3 R1, and RyR2. The inner and outer mitochondrial membrane proteins analyzed were COX1 and VDAC1, respectively; the characteristic MAM protein was ACSL-4. C, control, saline-injected; AβOs, AβOs-injected. The column at right indicates the ratios between band densities of each MAM protein (AβOs/control) normalized to b-tubulin protein levels.

Article Snippet: Primary antibodies: mouse monoclonal anti-RyR2 (MA3-916) was from Thermo Fisher Scientific (Waltham, MA, United States); rabbit monoclonal anti-RyR3 (AB9082) and rabbit polyclonal anti-IP 3 receptor type-1 (IP 3 R1) (AB5882) antibodies were from former Merck-Millipore (Darmstadt, Germany); rabbit polyclonal anti-ACSL4 (SAB2100035) and anti β-actin (A5316) were from Sigma-Aldrich (St. Louis, MI, United States); anti-VDAC (sc-390996), anti-Calnexin (sc-6465) and anti-COX4 (sc-69359) were from Santa Cruz Biotechnology (Dallas, TX, United States); rabbit monoclonal anti-β-amyloid (H31L21) was from Life Technologies (Waltham, MA, United States); rabbit c-Fos polyclonal antibody Ab-5 was from Oncogene (San Diego, CA, United States); Anti-Arc Polyclonal rabbit affinity purified antibody was from Synaptic System (Göttingen, Germany); ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technologies (Danvers, MA, United States).

Techniques: Derivative Assay, Injection, Western Blot, Isolation

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: A. Comparison of CD Ca 2+ binding site in IP 3 Rs. Overlay of rIP 3 R1 and hIP 3 R3 structures, highlighting the ARM2 and HD-IP 3 R1/CD-IP 3 R3 domains that form the Ca²⁺ binding site (outlined with a dashed line). B. Zoomed-in view of the Ca-CD site (rIP 3 R1 - 8EAR/grey; hIP 3 R3 - 6DRC/tan). The highly conserved R747-rIP 3 R1/R743-hIP 3 R3, E1127-rIP 3 R1/E1122-hIP 3 R3, E1130- rIP 3 R1/E1125-hIP 3 R3 responsible for coordination of Ca 2+ are labeled. Notably, Ca²⁺ has only been observed in the 6DRC structure. C. Structural alignment of the Ca-CD site between IP 3 R subtypes and through evolution. The Ca-CD site is not conserved in RyR. D. Overlay of rIP 3 R1 (green) and rabbit RyR1 (tan) structures (7M6A). The IP 3 R CD Ca 2+ binding residues are not conserved in rabbit RyR1 structure.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Comparison, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: Generation of stable cells in HEK-293 IP 3 R null-background A. A representative western blot depicting IP 3 R1 and GAPDH protein levels in HEK-3KO, HEK WT (HEK293), hR1 endo, stable hR1 exo 69, E1128A (clones #32 and #40), and E1131A (clones #6 and #18) cell lines. B. Scatter plot showing quantification of IP 3 R1 protein levels normalized to GAPDH. Data are presented as mean±s.e.m from n=4 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. *p<0.05, **p<0.01, ns; non-significant when compared to hR1 exo 69 cell line. C. Localization of IP 3 R1 in hR1 exo 69, E1128A #40, and E1131A #18 mutant stable cells. Scale bar, 10 µm.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Western Blot, Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: E1128A mutant stable cells exhibit enhanced sensitivity to IP 3 generating agonist compared to WT cells. Representative Ca 2+ traces showing increase in cytosolic Ca 2+ levels from A. hR1 exo, and B. E1128A cells in response to stimulation with 3, 10, and 30 μM CCh. The thicker black and red traces denote the average increase in cytosolic Ca 2+ from the population. Scatter plots comparing C. changes in amplitude (peak ratio – basal ratio: average of initial 20 ratio points) and D. percentage of responding cells (amplitude changes >0.1), from hR1 exo and E1128A cells in response to stimulation with 3, 10, and 30 μM CCh. Data are presented as mean±s.e.m from n=3 independent experiments. Statistical significance was determined by two-way ANOVA with Sidak test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns; non-significant. E. Pie-charts comparing cell response heterogeneity [non-responders, sustained/global Ca 2+ signals, and oscillating Ca 2+ signals (amplitude changes >0.05)] between hR1 exo 69 (n=64 cells) and E1128A #40 (n=67 cells) cell lines in response to stimulation with 3, 10, and 30 μM CCh.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: E1131A mutant stable cells exhibit enhanced sensitivity to IP 3 generating agonist compared to WT cells. Representative Ca 2+ traces showing increase in cytosolic Ca 2+ levels from A. hR1 exo and B. E1131A cells in response to stimulation with 0.3, 1, and 3 μM CCh. The thicker black and blue traces denote the average increase in cytosolic Ca 2+ from the population. Scatter plots comparing C. changes in amplitude (peak ratio – basal ratio: average of initial 20 ratio points) and D. percentage of responding cells (amplitude changes >0.1), from hR1 exo and E1131A cells in response to stimulation with 0.3, 1, and 3 μM CCh. Data are presented as mean±s.e.m from n=3 independent experiments. Statistical significance was determined by two-way ANOVA with Sidak test. *p<0.05, **p<0.01, and ****p<0.0001. E. Pie-charts comparing cell response heterogeneity [non-responders, sustained/global Ca 2+ signals, and oscillating Ca 2+ signals (amplitude changes >0.05)] between hR1 exo (n=58 cells) and E1131A (n=72 cells) cell lines in response to stimulation with 0.3, 1, and 3 μM CCh.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site Ca 2+ binding mutants exhibit spontaneous Ca 2+ puffs under basal conditions. A. Maximal intensity projections of Cal-520 fluorescence at 5 s intervals from hR1 exo, E1128A, and E1131A cell lines under basal resting conditions. Elementary Ca 2+ signals in E1128A, and E1131A cells are highlighted. B. Representative traces showing fluorescence changes from the center of a single puff site (1 × 1 μm) from hR1 exo (black), E1128A (red), and E1131A (blue) cell lines. C. Scatter plots summarizing number of puff sites/cell, number of puffs/cell, and average amplitudes of the Ca 2+ puffs are shown for hR1 exo, E1128A, and E1131A cells. Data are presented as mean±s.e.m from n=10 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. **p<0.01. D. Representative sweeps obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or mutant (E1128A, E1131A or CD DM) constructs without IP 3 in the presence of either 100 µM or 5 mM ATP and 1 µM [Ca 2+ ].

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Binding Assay, Fluorescence, Patch Clamp, Stable Transfection, Expressing, Mutagenesis, Construct

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site Ca 2+ binding mutants exhibit augmented Ca 2+ puffs upon uncaging ci-IP 3 . A. Maximal intensity projections of Cal-520 fluorescence at 5 s intervals from hR1 exo, E1128A, and E1131A cell lines upon uncaging 0.05 µM ci-IP 3 . Elementary Ca 2+ signals are highlighted. E. Representative traces showing fluorescence changes upon uncaging ci-IP 3 from the center of a single puff site (1 × 1 μm) from hR1 exo (black), E1128A (red), and E1131A (blue) cell lines. Individual Ca 2+ puffs from the boxed area highlighting longer decay/fall time for the CD site Ca 2+ binding mutants when compared to hR1 exo cells. Scatter plots summarizing B. number of puff sites/cell, C. number of puffs/cell, D. average amplitudes of the Ca 2+ puffs, and F. mean-rise (r) and -fall (f) times for the fluorescence to rise/decay to various levels (0%–100%) are shown. Data are presented as mean±s.e.m from n=10 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. **p<0.01, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: Generation of stable cells in DT-40 3KO background. A . A representative western blot depicting IP 3 R1 and GAPDH protein levels in DT40 exo R1 (clone #1), DT40 exo R1 E1128A, DT40 exo R1 E1131A, and DT40 exo R1 CD DM (E1128/1131A) cell lines. B. Scatter plot showing quantification of IP 3 R1 protein levels normalized to GAPDH. Data are presented as mean±s.e.m from n=3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s test. *p<0.05, **p<0.01, ns; non-significant when compared to DT40 exo R1 cell line. A comparison of intracellular global Ca 2+ signals (ΔF/F o ) between WT (DT40 R1) and CD DM cells following repeated photolysis of either C. 1 µM or D. 5 µM ci-IP 3 at indicated time points (arrows). Data are presented as mean±s.e.m from n=3 independent experiments.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Western Blot, Comparison

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site is not essential for Ca 2+ -dependent inactivation of IP 3 R1 at high [ATP]. A. Hill equation fit for pooled data obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or mutant (E1128A, E1131A or CD DM) constructs at indicated [Ca 2+ ] stimulated with 1 µM IP 3 in the presence of 5 mM ATP. B. Representative sweeps for the CD DM and hR1 at indicated [Ca 2+ ] in the presence of 1 µM IP 3 and 5 mM ATP are shown.

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Patch Clamp, Stable Transfection, Expressing, Mutagenesis, Construct

Journal: bioRxiv

Article Title: Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

doi: 10.1101/2024.08.16.608318

Figure Lengend Snippet: CD site is critical for Ca 2+ -dependent inactivation of IP 3 R1 at low [IP 3 ] and [ATP]. A. Hill equation fit for pooled data obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or mutant (E1128A, E1131A or CD DM) constructs at indicated [Ca 2+ ] stimulated with 1 µM IP 3 in the presence of 100 µM ATP. B. Representative sweeps showing higher activity from CD DM compared to R1 at inhibitory [Ca 2+ ]. C. Hill equation fit for pooled data obtained using “on-nucleus” patch clamp experiments from DT-40 3KO cells stably expressing either WT (R1) or CD DM mutant constructs at indicated [Ca 2+ ] stimulated with 10 µM IP 3 in the presence of 100 µM ATP. D. Representative sweeps showing higher activity from CD DM compared to R1 at inhibitory [Ca 2+ ].

Article Snippet: Following blocking, cells were incubated overnight at 4°C with primary antibody against IP 3 R1 (#ARC154, Antibody Research Corporation, 1:1000 dilution).

Techniques: Patch Clamp, Stable Transfection, Expressing, Mutagenesis, Construct, Activity Assay

Journal: bioRxiv

Article Title: Functional Communication between IP 3 R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

doi: 10.1101/2021.09.24.461545

Figure Lengend Snippet: ( A ) Overlay of mV-STIM2 (S2; Green) and IP 3 R1-mCherry (IP3R1; Red, expressed in STIM2-KI cells) with an enlarged image of demarcated area and line scan (position indicated on the enlarged overlay image). ( B ) Overlay images of S2 (top panel) and IP 3 R1-mCherry (IP 3 R1, lower panel) from 0 and 30s time points and line scans (position indicated on the enlarged overlay image). ( C ) Same cell as in A and B stimulated with 1μM CCh: overlay image of IP 3 R1 (Red) and S2 (Green) clusters and enlarged area (right). Line scan (below) show positions of the two proteins in two clusters (indicated by green arrow). ( D ) STIM2-KI cells showing S2 (upper panel) or cells with knockdown of all IP 3 R isoforms (lower panel). In each case, S2 fluorescence in unstimulated and 1µM CCh- stimulated cells are shown. ( E ) Increase in S2 cluster (mean intensity, top, and relative cluster number,bottom, in control (black trace) and IP 3 Rs knockdown cells (red trace)). ( F ) STIM2-KI cells expressing mCherry-ER3 showing S2 (Green) and mCherry-ER3 (ER, Red) in control (upper panels) and siIP 3 Rs-treated (lower panels) without stimulation. ( G ) Wild-type HEK293 transfected with IP 3 R1 or IP 3 R1+siE-Syts2/3. ( H ) Overlay images of S2 clusters in STIM2-KI cells alone, or with si-Esyt2/3 or siIP 3 Rs treatment from 0 and 1 min time points (0/1m). Line scan show overlapping peaks of both time points under basal condition. All TIRFM images show representative cells from at least 3 experiments. Scale bars, 5µm.

Article Snippet: Proteins of interest were immunoblotted using anti-IP 3 R1 and anti- Myc, anti-STIM2 (Cell Signaling Technologies, Danvers, MA), anti-β-actin (Abcam, Cambridge, MA) antibodies.

Techniques: Fluorescence, Expressing, Transfection

Journal: bioRxiv

Article Title: Functional Communication between IP 3 R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

doi: 10.1101/2021.09.24.461545

Figure Lengend Snippet: ( A ) mV-STIM2 clusters before and after 5µM forskolin (FSK) stimulation (top panel). Ooverlay images of S1 at 0 and 1 min (0/1m) and 3 and 4 min (3/4m) time points (bottom panel). ( B ) Increase of mV-S2 mean fluorescence intensity (MI) and relative number (Cluster #) ( C ) in response to FSK treatment (added at 1 min time point, dotted line). ( D ) HEK293 cells expressing TK-YFP-STIM2 (TK-S2) before and after stimulation with FSK. ( E ) IP 3 R-TKO cells (lack all three IP 3 R sub-types), treated with siE-Syts2/3 and expressing TK-S2, before and after stimulation with 5µM FSK and 25µM cyclopiazonic acid (CPA). Bar graphs showing number (Puncta #) and mean intensity intensity of TK-S2 clusters in the three conditions shown in the images ( n =8). Statistical tests were done using ANOVA with the significance presented as not significant (n.s.; P > 0.05) and significant (* P < 0.05). ( F ) STIM2- KI cells expressing ER-Lar-GECO1: S2 (upper panel) and ER-Lar-GECO1 (lower panel) under basal (unstimulated) and stimulated with FSK (5µM). Enlargements of the region marked by a square are shown for visible camparisions. ( G ) Bar graph shows change in relative mean fluorescence intensity (MI) at the 5 min time point compared to basal (time point 1 min). Data is from 3 experiments and n=43 immobile clusters). ( H ) Line graphs showing whole cell intensity of ER- Lar-GECO1 in wild-type (WT), STIM2-KI (S2KI) and IP 3 R-TKO cells, and S2 fluorescence only in S2KI expressing Lar-GECO1. Addition of 5µM FSK is indicated by the black arrows. All TIRFM images show representative cells from at least 3 experiments. Scale bars, 5µm.

Article Snippet: Proteins of interest were immunoblotted using anti-IP 3 R1 and anti- Myc, anti-STIM2 (Cell Signaling Technologies, Danvers, MA), anti-β-actin (Abcam, Cambridge, MA) antibodies.

Techniques: Fluorescence, Expressing

Journal: bioRxiv

Article Title: Functional Communication between IP 3 R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

doi: 10.1101/2021.09.24.461545

Figure Lengend Snippet: ( A ) Immunoprecipitation (IP) of IP 3 R1 from HEK293 cells expressing Myc- STIM1 and Myc-STIM2. Immunoblotting (IB) was done using the anti-IP 3 R1 and anti-Myc antibody, respectively, for IP fractions (top and second blots) and input lysates (third and bottom blots). IB antibodies are shown next to each blot. ( B ) Reverse IP using the same cell lysate as in ( A ), but using anti-Myc antibody to pull down STIM1 and STIM2. The blots are representative of data obtained from 2 experiments. ( C ) HEK293 and IP 3 R-TKO cells expressing genetically encoded Ca 2+ indicators, ER- Lar-GECO1 in basal (unstimulated condition), FSK (cells stimulated with Forskolin, 5µM). Enlargements of the region marked by square are shown for visible comparison. All images are TIRF micrographs and representative of data obtained from >3 experiments. FSK was added at the 2 min time point for all experiments. Scale bar, 5µm. ( D ) Bar graphs showing ER- Lar-GECO1 fluorescence (F/F 0 ) before and after stimulation with 5µM FSK in wild-type (WT) HEK293, IP 3 R-TKO, and STIM2-KI (S2KI) cells. The graph shows averaged data (mean ± SEM) obtained from n = 16 Cells (WT), 13 Cells (IP 3 R-TKO), and 6 Cells (S2KI).

Article Snippet: Proteins of interest were immunoblotted using anti-IP 3 R1 and anti- Myc, anti-STIM2 (Cell Signaling Technologies, Danvers, MA), anti-β-actin (Abcam, Cambridge, MA) antibodies.

Techniques: Immunoprecipitation, Expressing, Western Blot, Fluorescence