ip 3 r2  (Pocono Rabbit Farm)

Journal: Cell reports

Article Title: Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14

doi: 10.1016/j.celrep.2023.113437

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Taqman Assay for IP 3 R2 (ITPR2) , ThermoFisher , Hs00181916_m1.

Techniques: Virus, Recombinant, Reverse Transcription, DC Protein Assay, Western Blot, CRISPR, Control, Expressing, TaqMan Assay, shRNA, Software

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) Confocal images of hNPCs (passage 6) stained for DAPI and neural stem cell proteins: Pax6 and Ki67 (proliferation marker). Scale bars, 50 μm. ( B ) WB for IP 3 R1 of hNPCs expressing non-silencing (NS) or IP 3 R1-shRNA. ( C ) Summary results (mean ±s.d., n=3) show IP 3 R1 expression relative to actin. ** p < 0.01, Student’s t -test with unequal variances. ( D ) Changes in [Ca 2+ ] c evoked by thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then restoration of extracellular Ca 2+ (2 mM) in hNPCs expressing NS or IP 3 R1-shRNA. Mean ± s.e.m. from hree independent experiments, each with four replicates that together included 100–254 cells. Inset shows the target of Tg. ( E–G ) Summary results (individual cells, median (bar), 25th and 75th percentiles (box) and mean (circle)) show Ca 2+ signals evoked by Tg or Ca 2+ restoration ( E ), rate of Ca 2+ entry ( F ) and resting [Ca 2+ ] c ( G ). *** p < 0.001, Mann-Whitney U-test. ( H ) Changes in [Ca 2+ ] c evoked by Tg (10 µM) in Ca 2+ -free HBSS and after restoring extracellular Ca 2+ (2 mM) in neurons (differentiated hNPCs) expressing NS or IP 3 R1-shRNA. Mean ± s.e.m. from three experiments with ~200 cells. ( I,J ) Summary results (presented as in E-G) show Ca 2+ signals evoked by Tg or Ca 2+ restoration ( I ) and rate of Ca 2+ entry ( J ). *** p < 0.001. Mann-Whitney U-test. See also . Source data in . Figure 1—source data 1. Loss of IP 3 R1 attenuates SOCE in human neural stem cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Staining, Marker, Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) WB for IP R1-3 of SH-SY5Y cells expressing non-silencing (NS) or IP R1-shRNA. ( B ) Summary results (mean ± s.d., n=4) show IP R expression relative to actin normalized to control NS cells. ** p < 0.01, Student’s t -test with unequal variances. ( C ) Ca 2+ signals evoked by carbachol (CCh, 3 µM) in SH-SY5Y cells expressing NS or IP R1-shRNA. Mean ± s.e.m. from three experiments with 70–90 cells. ( D ) Summary results show peak changes in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by CCh. *** p < 0.001, Mann-Whitney U-test. ( E ) Ca 2+ signals evoked by thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then after restoration of extracellular Ca 2+ (2 mM) in cells expressing NS or IP R1-shRNA. Mean ± s.e.m. from three experiments with ~50 cells. ( F, G ) Summary results (individual cells, mean ± s.e.m., n=3, ~50 cells) show peak changes in [Ca 2+ ] c evoked by Ca 2+ restoration (Δ[Ca 2+ ] c ) ( F ) and rate of Ca 2+ entry ( G ). *** p < 0.001, Mann-Whitney U-test. ( H ) Ca 2+ signals evoked by Tg and then Ca 2+ restoration in cells expressing NS-shRNA, or IP R1-shRNA alone or with IP R1 or IP R3. Traces show mean ± s.e.m. (50–115 cells from three experiments). ( I, J ) Summary results (mean ± s.e.m, 50–115 cells from three experiments) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration ( I ) and rates of Ca 2+ entry ( J ) evoked by restoring extracellular Ca 2+ . ( K ) Effects of thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then after Ca 2+ restoration (2 mM) in cells expressing IP R1-shRNA alone or with IP R1 or mCh-STIM1. Traces show mean ± s.e.m. (100–150 cells from three experiments). ( L, M ) Summary results (mean ± s.e.m.) show peak increase in [Ca 2+ ] c after Ca 2+ restoration (Δ[Ca 2+ ] c ) ( L ) and rate of Ca 2+ entry ( M ). Different letters indicate significant differences (panels I , J, L, M), p <0.001, one-way ANOVA with pair-wise Tukey’s test. See also – . Source data in . Figure 2—source data 1. Loss of IP 3 R1 attenuates SOCE in SH-SY5Y cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) SOCE is activated when loss of Ca 2+ from the ER through IP 3 Rs activates STIM1 ( i ). Our results suggest an additional role for IP 3 Rs (ii). ( B ) SH-SY5Y cells expressing IP 3 R1-shRNA alone or with IP 3 R1 or IP 3 R1 DA were stimulated with thapsigargin (Tg, 1 µM) in Ca 2+ -free HBSS before restoring extracellular Ca 2+ (2 mM). Traces show mean ± s.e.m, for 100–150 cells from three experiments. ( C ) Cells expressing IP 3 R1-shRNA and IP 3 R1 DA were treated with NS-siRNA or Orai1-siRNA before measuring Tg-evoked Ca 2+ entry. Traces show mean ± s.e.m. for 85–100 cells from three experiments. ( D ) Summary results (mean ± s.e.m.) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration. ( E ) Tg-evoked Ca 2+ entry in cells expressing IP 3 R1-shRNA with IP 3 R1, IP 3 R1 RQ or IP 3 R1 RQ/KQ . Traces show mean ± s.e.m, for 90–150 cells from three experiments. ( F ) Summary results (mean ± s.e.m.) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration. Different letter codes (panels D , F ) indicate significantly different values, p<0.001, for multiple comparison one-way ANOVA and pair-wise Tukey’s test and for two genotype comparison Mann Whitney U-test. See also . Source data in . Figure 3—source data 1. Regulation of SOCE by IP 3 R requires IP 3 binding but not a functional pPore in SH-SY5Y cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, MANN-WHITNEY, Binding Assay, Functional Assay

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A, B ) SH-SY5Y cells expressing IP 3 R1-shRNA alone ( A ) or with IP 3 R1 DA ( B ) were treated with a low concentration of CPA (2 µM) in Ca 2+ -free HBSS to partially deplete the ER of Ca 2+ and sub-maximally activate SOCE (see ). Carbachol (CCh, 1 µM) was then added to stimulate IP 3 formation through muscarinic receptors, and extracellular Ca 2+ (2 mM) was then restored. Traces (mean ± s.e.m of 68–130 cells from three experiments) show responses with and without the CCh addition. ( C ) Summary results show the peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) after addition of CCh (CCh-induced Ca 2+ release) and then after restoring extracellular Ca 2+ (SOCE). ( D–F ) SH-SY5Y cells wild type (WT) ( D ) and expressing NS-shRNA ( E ) or IP 3 R1-shRNA ( F ) were treated with YM-254890 (YM, 1 µM, 5 min) in Ca 2+ -free HBSS to inhibit Gαq and then with thapsigargin (Tg, 1 µM) before restoring extracellular Ca 2+ (2 mM). Traces show mean ± s.e.m of ~120 cells from three experiments. ( G–I ) Similar analyses of HEK cells. Summary results (mean ± s.e.m, 50–100 cells from three experiments) are shown in ( I ). Different letter codes (panels C and I) indicate significantly different values within the store Ca 2+ release or SOCE groups, p<0.001, one-way ANOVA and pair-wise Tukey’s test. See also . Source data in . Figure 4—source data 1. Receptor-regulated IP 3 production stimulates SOCE in cells with empty Ca 2+ stores and expressing pore-dead IP 3 R.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, Concentration Assay

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A–E ) PLA analyses of interactions between STIM1 and Orai1 in SH-SY5Y cells expressing NS-shRNA ( A ) or IP 3 R1-shRNA alone ( B ) or with IP 3 R1 ( C ), IP 3 R1 DA ( D ) or IP 3 R1 RQ/KQ ( E ). Confocal images are shown for control cells or after treatment with thapsigargin (Tg, 1 µM) in Ca 2+ -free HBSS. PLA reaction product is red, and nuclei are stained with DAPI (blue). Scale bars, 5 µm. Summary results show the surface area of the PLA spots for 8–10 cells from two independent analyses. Individual values, median (bar) and 25th and 75th percentiles (box). *** p < 0.001, Student’s t -test with unequal variances. See also . Source data in . Figure 5—source data 1. IP 3 Rs promote interaction of STIM1 with Orai1.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, Staining

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A–B ) Representative TIRF images of mVenus STIM1 co-transfected with either wild type mcherry-rat IP 3 R1 ( A ) or IP 3 R1 RQ/KQ (ligand binding mutant), ( B ) in wild type SH-SY5Y cells before (Basal) and after CPA induced store depletion (CPA treated) at 4 min and 7 min. On the right are shown RGB profile plots of STIM1 (green) and IP 3 R1, wild type or mutant (magenta) corresponding to the rectangular selections (Cell 1 and Cell 2). Scale bar is 10 µm.( C–D ) Changes in number of IP 3 R1 ( C ) and STIM1 ( D ) puncta upon CPA-induced store depletion over a period of 10 min in the indicated genotypes. Mean ± s.e.m from seven cells from n=6 independent experiments. ( E ) Summary result (mean ± s.e.m) showing the change in the number of maximum STIM1 puncta formed after CPA-induced store depletion in the indicated genotypes. Mean ± s.e.m. of seven cells from n=6 independent experiments. Different letters indicate significant differences, p<0.05, Mann-Whitney U-test. See also . Source data in . Figure 6—source data 1. Ligand-bound IP 3 R1 supports SOCE-dependent STIM1 movement to ER-PM contact sites.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Transfection, Ligand Binding Assay, Mutagenesis, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) SOCE is activated when loss of Ca 2+ from the ER, usually mediated by opening of IP 3 Rs when they bind IP 3 , causes STIM to unfurl cytosolic domains (2). The exposed cytosolic domains of STIM1 reach across a narrow gap between the ER and PM at a MCS to interact with PIP 2 and Orai1 in the PM. Binding of STIM1 to Orai1 causes pore opening, and SOCE then occurs through the open Orai1 channel. We show that IP 3 Rs when they bind IP 3 also facilitate interactions between Orai1 and STIM, perhaps by stabilizing the MCS (1). Receptors that stimulate IP 3 formation thereby promote both activation of STIM (by emptying Ca 2+ stores) and independently promote interaction of active STIM1 with Orai1. ( B ) Other mechanisms, including ryanodine receptors (RyR), can also release Ca 2+ from the ER. We suggest that convergent regulation of SOCE by IP 3 R with bound IP 3 allows receptors that stimulate IP 3 formation to selectively control SOCE.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Binding Assay, Activation Assay