Journal: BMC Physiology

Article Title: Differential role of STIM1 and STIM2 during transient inward ( T in ) current generation and the maturation process in the Xenopus oocyte

doi: 10.1186/s12899-014-0009-x

Figure Lengend Snippet: STIM expression in the Xenopus oocyte and its downregulation by as-STIM injection. A) shows the RT-PCR amplification of products that corresponded to the size expected for either stim1 or stim2 in native oocytes (CNT); the corresponding amplicons were absent in oocytes from the same batch that had been injected with either as-STIM1 or as-STIM2 48 h before the assay. The rps2 amplicon indicates the reaction efficiency, and -RT and H 2 O lanes correspond to negative controls, either RNA without RT, or to the reaction mix without a cDNA template, respectively. B) STIM1 and STIM2 were identified by Western blot analysis in protein extracts from oocytes (Oo) or mouse brain (MB, positive control) using either NH-STIM1 (left panel) or COOH-STIM2 (right panel) as antibody. C) A similar analysis as in B was made for batches of oocytes injected with H 2 O as control (CNT), or with as-STIM1 or as-STIM2 48 h before the protein extraction, in which cases proteins were eliminated. (in all cases 10 oocytes per condition).

Article Snippet: The antibody denoted NH-STIM1 (Alomone, Jerusalem, Israel) was directed against a region of the amino-terminus of the STIM1 protein, and the antibodies denoted NH-STIM2 (Alomone, Jerusalem, Israel) and COOH-STIM2 (ProSci Inc., Poway CA, USA) were against the amino and carboxy termini, respectively, of STIM2.

Techniques: Expressing, Injection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Positive Control, Protein Extraction

Journal: BMC Physiology

Article Title: Differential role of STIM1 and STIM2 during transient inward ( T in ) current generation and the maturation process in the Xenopus oocyte

doi: 10.1186/s12899-014-0009-x

Figure Lengend Snippet: Knockdown of STIM expression in oocytes co-injected with GPCR mRNA. A) RT-PCR amplification of stim1 , stim2 , or rps2 in batches of oocytes injected with H 2 O (CNT) or with cRNA (50 ng per oocyte) coding for either P2Y8 or M1 GPCR. In oocytes co-injected with as-STIM1 or as-STIM2 (50 ng per oocyte) together with P2Y8 or M1 cRNA, the corresponding STIM amplicon was downregulated. Control reactions illustrate specificity; rps2 amplicons are positive controls, and -RT and H 2 O lanes show negative controls. B) Similar groups of oocytes as in A) were assayed using the Western blot technique; in this case oocytes from the same donor injected with one GPCR mRNA (P2Y8 or M1) alone, or co-injected with as-STIM1, were tested with NH-STIM1, while as-STIM2-injected oocytes were probed with COOH-STIM2. In both as-STIM groups SERCA was used as gel-loading control. C) The graph shows the densitometric analysis of bands, summarizing the results obtained in different preparations of 10 oocytes per group and repeated in 3–5 frogs. Both PCR products and bands detected by Western blot (WB) were analyzed for batches of oocytes injected with H 2 O (CNT) or with either 50 ng as-STIM1 or as-STIM2 alone (native group). Similar analysis was made for batches of control oocytes injected with P2Y8 or M1 cRNA alone, and oocytes from the same frogs co-injected with either as-STIM or as-STIM together with the GPCR cRNA. Optical density units (ODU) for each band were normalized against the value obtained in the corresponding CNT conditions (*p < 0.01).

Article Snippet: The antibody denoted NH-STIM1 (Alomone, Jerusalem, Israel) was directed against a region of the amino-terminus of the STIM1 protein, and the antibodies denoted NH-STIM2 (Alomone, Jerusalem, Israel) and COOH-STIM2 (ProSci Inc., Poway CA, USA) were against the amino and carboxy termini, respectively, of STIM2.

Techniques: Expressing, Injection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot

Journal: BMC Physiology

Article Title: Differential role of STIM1 and STIM2 during transient inward ( T in ) current generation and the maturation process in the Xenopus oocyte

doi: 10.1186/s12899-014-0009-x

Figure Lengend Snippet: I osc and T in responses activated by agonist stimulation. A) Strength of I osc elicited by first agonist application did not change by knockdown of STIM1 or STIM2, compared with that obtained in CNT oocytes; top traces are typical responses elicited by ACh, similar responses were obtained by FBS or ATP applications, and the graph shows the average I osc responses obtained in oocytes held at −60 mV. B) Record illustrating the activation of T in current obtained in an oocyte expressing the M1 receptor by a single ACh (100 μM) application for 40 s (acute protocol). Oocytes were held at −10 mV while being superfused with NR solution and stepped to −100 mV for 4 s every 40 s; sudden hyperpolarization generated T in current responses that follow consistent kinetics with a peak amplitude response at 280–360 s (c); after that the response was washed out with a similar time course. C) Shows the T in current during the steps from −10 to −100 mV indicated with letters in panel B) . D) A similar T in current response elicited in an oocyte from the same frog that was pre-incubated with 1 μM ACh for 4 h (long-lasting protocol), then monitored with the same electrical recording parameters and stimulated with 100 μM ACh. E) Shows the T in responses indicated with the same letters as in D) . In this protocol T in current was consistently activated from the beginning of the record, and a transient inhibition of the response was noted during application of the agonist ( b) ; after that, T in recovered and remained fully activated for a long period of time. Similar responses were obtained using oocytes expressing P2Y receptors and stimulating with ATP.

Article Snippet: The antibody denoted NH-STIM1 (Alomone, Jerusalem, Israel) was directed against a region of the amino-terminus of the STIM1 protein, and the antibodies denoted NH-STIM2 (Alomone, Jerusalem, Israel) and COOH-STIM2 (ProSci Inc., Poway CA, USA) were against the amino and carboxy termini, respectively, of STIM2.

Techniques: Activation Assay, Expressing, Generated, Incubation, Inhibition

Journal: BMC Physiology

Article Title: Differential role of STIM1 and STIM2 during transient inward ( T in ) current generation and the maturation process in the Xenopus oocyte

doi: 10.1186/s12899-014-0009-x

Figure Lengend Snippet: Specific STIM knockdown by oocyte injection of as-STIM differentially decreased the T in current. A) Oocytes induced to express M1, P2Y8, or P2Y2 receptors were stimulated with either ACh or ATP (100 μM), and LPAR in native oocytes were stimulated by FBS (1:1000 dilution); the resulting T in currents (CNT, gray areas) were compared with the T in obtained in oocytes from the corresponding group that were also injected with 50 ng as-STIM1 (superimposed black traces); all responses were monitored 48–72 h after oocyte injection. B) The graph shows the results obtained using the different experimental conditions illustrated in A) . C) In a set of experiments similar to those shown in A) , T in currents were monitored, and the peak amplitudes of non-injected CNT oocytes were compared with those of oocytes injected (48–72 h before recording) with 50 ng as-STIM2 and stimulated with the agonists. D) The graph shows the results obtained using the different experimental conditions illustrated in C) . Bars correspond to the mean (± SEM) of the T in peak amplitude of 10–15 oocytes from 5–6 frogs (*p < 0.01, as-STIM vs. CNT).

Article Snippet: The antibody denoted NH-STIM1 (Alomone, Jerusalem, Israel) was directed against a region of the amino-terminus of the STIM1 protein, and the antibodies denoted NH-STIM2 (Alomone, Jerusalem, Israel) and COOH-STIM2 (ProSci Inc., Poway CA, USA) were against the amino and carboxy termini, respectively, of STIM2.

Techniques: Injection

Journal: BMC Physiology

Article Title: Differential role of STIM1 and STIM2 during transient inward ( T in ) current generation and the maturation process in the Xenopus oocyte

doi: 10.1186/s12899-014-0009-x

Figure Lengend Snippet: Oocyte injection with COOH-STIM2 antibody produced a strong potentiation of T in current response. A) T in current responses were monitored in two conditions: non-loaded oocytes (CNT) and oocytes loaded with COOH-STIM2 antibody (ab-loaded). T in responses were elicited by ACh, FBS, or ATP application, depending on the receptor to be stimulated. In all cases, a strong potentiation of the response was observed in ab-loaded oocytes. B) Oocytes stimulated by ACh (M1) loaded with denatured COOH-STIM2 had control-like responses, while NH-STIM2 or NH-STIM1 loading did not produce T in potentiation. C) The graph shows the results obtained using the different experimental conditions illustrated in A and B ; each bar corresponds to the mean (± SEM) of the T in peak amplitude normalized against the CNT current of 10–15 oocytes from 3–6 frogs (*p < 0.01).

Article Snippet: The antibody denoted NH-STIM1 (Alomone, Jerusalem, Israel) was directed against a region of the amino-terminus of the STIM1 protein, and the antibodies denoted NH-STIM2 (Alomone, Jerusalem, Israel) and COOH-STIM2 (ProSci Inc., Poway CA, USA) were against the amino and carboxy termini, respectively, of STIM2.

Techniques: Injection, Produced

Journal: BMC Physiology

Article Title: Differential role of STIM1 and STIM2 during transient inward ( T in ) current generation and the maturation process in the Xenopus oocyte

doi: 10.1186/s12899-014-0009-x

Figure Lengend Snippet: Effect of as-STIM2 on GVBD and oocyte membrane characteristics during maturation induced by progesterone. A) The maturation process promoted by progesterone (10 μM) was analyzed in uninjected oocytes, or in oocytes injected 72 h prior to the assay with either as-STIM1 or as-STIM2, and compared with control oocytes in the absence of progesterone. GVBD was quantified after 8–12 h in presence of progesterone (10 oocytes per group, repeated using 3 different frogs) and is normalized against the value observed in uninjected oocytes. B) Resting membrane potential was monitored 8–12 h after addition of progesterone in the same groups of oocytes (n = 3-5, repeated in 3 frogs) as in A) . C) The input membrane resistance (Rϕ) was estimated over the range from −80 to −20 mV in the different oocyte groups treated in the same conditions. Control groups, without progesterone, included both uninjected and antisense-injected oocytes. In all cases, values for as-STIM2-injected groups were different from as-STIM1-injected or uninjected groups (*p < 0.01).

Article Snippet: The antibody denoted NH-STIM1 (Alomone, Jerusalem, Israel) was directed against a region of the amino-terminus of the STIM1 protein, and the antibodies denoted NH-STIM2 (Alomone, Jerusalem, Israel) and COOH-STIM2 (ProSci Inc., Poway CA, USA) were against the amino and carboxy termini, respectively, of STIM2.

Techniques: Injection

Journal: Cell Death Discovery

Article Title: Abrogation of store-operated Ca 2+ entry protects against crystal-induced ER stress in human proximal tubular cells

doi: 10.1038/s41420-019-0203-5

Figure Lengend Snippet: Crystal internalization induces upregulation of STIM and ORAI channel expression. Control (noncrystal) CaP, CaOx, and CaP + CaOx (mixed) crystals were introduced into HK2 cells for 24 h. a mRNA expression levels of STIM1, 2, and ORAI channels were analyzed by PCR. ORAI and STIM expression is mediated by SOCE in HK2 cells. HK2 cells were incubated and representative blots were obtained, relative mRNA expression was quantified and represented as bar diagram. b To confirm knockdown following siRNA (10 nM), b STIM1; c STIM2; and d ORAI3 transcript were determined by PCR, representative blots were obtained and quantified as bar diagrams. e To ascertain the role of STIM12 and ORAI3 channel in crystal internalization, STIM1, 2, and ORAI3 expression was knocked down using siRNA, cells were then introduced with or without CaP, CaOx, and mixed (CaP + CaOx) crystals for 24 h. Following crystal internalization, cells were loaded with fura-2AM and Ca 2+ influx was determined and represented as d [Ca 2+ ] rise and e Ca 2+ influx was determined and represented as reduction in [Ca 2+ ] response. Two-tailed t -test was used for statistical comparison. Statistically significant differences are indicated (mean ± SEM) from three different experiments. Levels of significance are indicated as * p < 0.05; ** p < 0.01 as shown in the bar diagrams

Article Snippet: HK2 cells were transfected with (10 nM) siRNA against STIM1 (Santa Cruz Biotechnology, Santa Cruz CA; sc-76589), STIM2 (Santa Cruz sc-76591), ORAI3 (Santa Cruz sc-76005), and scrambled siRNA-A (Santa Cruz sc-37007) as negative control.

Techniques: Expressing, Control, Incubation, Knockdown, Two Tailed Test, Comparison

Journal: Cell Death Discovery

Article Title: Abrogation of store-operated Ca 2+ entry protects against crystal-induced ER stress in human proximal tubular cells

doi: 10.1038/s41420-019-0203-5

Figure Lengend Snippet: Control (noncrystal) CaP, CaOx, and CaP + CaOx (mixed) crystals were introduced into HK2 cells for 24 h. a mRNA levels of ER stress genes ERN (ERN1), CLDN (CLDN1), and GRP78 were analyzed by PCR and representative blots were obtained and quantified. STIM1, STIM2, and ORAI3 expression was knocked down using siRNA transfection (10 nM). Following transfection, b CaP; c CaOx, and d Mixed crystals were introduced into HK2 cells and mRNA levels of ER stress genes ERN1, CLDN1, and GRP78 were analyzed by PCR and representative blots were obtained and quantified. Two-tailed t -test was used for statistical comparison. Statistically significant differences are indicated (mean ± SEM) from three different experiments. Levels of significance are indicated as * p < 0.05; ** p < 0.01 as shown in the bar diagrams

Article Snippet: HK2 cells were transfected with (10 nM) siRNA against STIM1 (Santa Cruz Biotechnology, Santa Cruz CA; sc-76589), STIM2 (Santa Cruz sc-76591), ORAI3 (Santa Cruz sc-76005), and scrambled siRNA-A (Santa Cruz sc-37007) as negative control.

Techniques: Control, Expressing, Transfection, Two Tailed Test, Comparison

Journal: Molecular Cell

Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

doi: 10.1016/j.molcel.2021.10.025

Figure Lengend Snippet: RHBDL2 prevents inappropriate CRAC channel activation in resting cells (A) Scheme of the hOrai1-K-GECO and Cyto-G-GECO reporter assay for spontaneous CRAC channel activity. K-GECO fluorescence is a measure of CRAC channel activity; G-GECO fluorescence is a measure of global cytoplasmic Ca 2+ fluctuations. (B) Four representative line traces of K-GECO fluorescence in single HaCaT cells treated with control siRNA or RHBDL2 siRNA. Spontaneous CRAC channel activation is defined as a >20% increase in the peak amplitude (ΔF/F0). (C) Quantification of spontaneous CRAC channel activity, defined as a >20% increase above the baseline K-GECO fluorescence (see Figure S4 C). Quantification of the percentage of cells displaying spontaneous CRAC channel activity (38 individual cells analyzed in control, 45 individual cells analyzed in RHBDL2 siRNA cells), and the K-GECO fluorescence spike frequency (number of spontaneous CRAC channel activity events over 10 min, relative to the number of cells). (D) TaqMan assay for TNF-alpha in HaCaT cells treated with control or RHBDL2 siRNAs for 48 h. Cyclosporin A (1 μm) was added for the final 24 h. Error bars represent RQ standard error. Each bar represents one of at least four biological replicates. (E) A scheme of the Stim1-BirA experiment in (F) and (G), illustrating the biotinylation of V5-hOrai1 (orange) by Stim1-BirA ∗ (green) at PM-ER contact sites, and the consequence of RHBDL2 (blue) activity. Biotin is indicated by green dots. For simplicity, Stim1 and Orai1 are illustrated as monomeric. (F and G) Western blots of neutravidin agarose-based biotin preparations (in F and G, upper) and total cell lysates (in G, lower) from wild-type (WT) or RHBDL2 knockout (R2 KO) HaCaT cells. The expression of V5-hOrai1 and Stim1-BirA ∗ was induced with doxycycline (DOX; 250 μg/mL final) for 96 h in the presence of 50 μm biotin. Six hours prior to lysis, bafilomycin A1 (BAF; 100 nm final) was added to block lysosomal degradation. In (G), cells were treated twice with Stim2 siRNAs for 96 h prior to lysis. Blots were probed for the N-terminal or C-terminal epitopes recognized by V5 and O1 antibodies, respectively. Stim1 and Stim1-BirA ∗ were probed for using an anti-Stim1 antibody. Different full-length forms of Orai1 and their cleavage products are indicated by the blue arrowheads. (H) Western blots of HaCaT lysates after treatment with control and RHBDL2 siRNA for 72 h, and when used, Stim2 siRNA for 96 h, labeled for endogenous Orai1, Stim1, and beta-actin. Full-length Orai1 (FL) and Orai1β (FLβ) are indicated by arrowheads. (I) Quantification of the fold change in Orai1 protein abundance, from three independent experiments performed as in (H). Error bars represent SEM.

Article Snippet: The Applied Biosystems TaqMan probes, all purchased through Life Technologies, were as follows: RHBDL2 (Hs00983274_m1), TNF alpha (Hs00174128_m1), IL-6 (Hs00985639_m1), Stim2 (Hs00957788_m1) and GAPDH (Hs02786624_g1).

Techniques: Activation Assay, Reporter Assay, Activity Assay, Fluorescence, Control, TaqMan Assay, Western Blot, Knock-Out, Expressing, Lysis, Blocking Assay, Labeling, Quantitative Proteomics

Journal: Molecular Cell

Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

doi: 10.1016/j.molcel.2021.10.025

Figure Lengend Snippet:

Article Snippet: The Applied Biosystems TaqMan probes, all purchased through Life Technologies, were as follows: RHBDL2 (Hs00983274_m1), TNF alpha (Hs00174128_m1), IL-6 (Hs00985639_m1), Stim2 (Hs00957788_m1) and GAPDH (Hs02786624_g1).

Techniques: Virus, Recombinant, Gene Expression, Control, Negative Control, shRNA, Mutagenesis, Plasmid Preparation, Software

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 2. Cellular level of STIM1 protein is elevated and that of STIM2 is decreased in MEF DKO cells. (A,D) Representative immunoblots of protein extracts from B cell lines developed with anti-STIM1 and anti-STIM2 antibodies and results of densitometric analysis of five independent experiments. (B,C) Results of RT-PCR quantification of STIM1 and STIM2 mRNA levels in MEF cell lines derived from wt, PS1−/−, PS2−/−or DKO (lacking both PS1 and PS2) animals.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 3. CCE is attenuated but STIM proteins' levels are not affected by overexpression of PS1 FAD mutants in HEK293 cells. (A) Averaged traces obtained by ratiometric Fura-2 analysis of HEK293 cell lines overexpressing wt PS1 and PS1 P117R and PS1 E318G mutants. (B) Results of quantification of CCE in analyzed HEK293 cell lines (⁎⁎⁎Pb0,001). (C) Representative immunoblot of protein extracts from HEK293 cell lines developed with anti-STIM1 and anti-STIM2 (D) antibodies and results of densitometric analysis of three independent experiments.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Over Expression, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 5. STIM2 mRNA and protein levels are decreased in B lymphocytes isolated from FAD patients. (A,C) Results of RT-PCR quantification of STIM1 and STIM2 mRNA levels in cell populations from healthy controls and FAD patients. (B,D) Representative immunoblot of protein extracts from B lymphocytes lines developed with anti-STIM1 and anti-STIM2 (D) antibodies and results of densitometric analysis of three independent experiments.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Annals of Clinical and Translational Neurology

Article Title: A familial t(4;8) translocation segregates with epilepsy and migraine with aura

doi: 10.1002/acn3.51040

Figure Lengend Snippet: (A) Array‐CGH ratio profiles of proband III‐2. Chromosomes 4 and 8 ideograms showing the 27.3 Mb 4p terminal duplication, the 3.8 Mb 8p terminal deletion, and magnifications of the breakpoint regions showing gene content and genomic positions, according to the genome build CGRh37/hg19. (B) Array‐CGH profiles of two rare CNVs identified in III‐1 genome. 1q43 deletion, identified in III‐1 and her mother (II‐1), located about 112 kb from the 3’UTR of RGS7 gene, and 5q12.3 deletion, partially involving the RGS7BP gene, identified in III‐1, III‐2, and their father (II‐2). Gene content and genomic positions are shown, according to the genome build CGRh37/hg19. (C) FISH analysis of translocation breakpoints. FISH with BAC clone CTD‐322006 (I,II), which spans the translocation bkp at 8p23.3, yields hybridization signal on chromosome 8 and signals of diminished intensity on both derivative chromosomes in II‐2 (I), while in III‐2 (II) the probe produces a signal on chromosome 8 and a signal of diminished intensity on der(8). FISH with BAC clone CTD‐2182P13 (III,IV), partially covering the STIM2 gene sequence at 4p15.2, yields hybridization signals of equal intensity on chromosomes 4 and der(8) in II‐2 (III), but produces three signals of the same intensity on both chromosomes 4 and on der(8) in the proband III‐2 (IV). (D) Physical map of the genomic regions involved in the bkps. 4p15.2‐15.1 region showing the mapped genes, the position of the 4p translocation bkp and its distance from the 3’UTR of STIM2 gene, and the probe CTD‐2182P13used for FISH analysis. 8p23.2 region showing the position of the 8p translocation bkp disrupting the CSMD1 gene at IVS3 and the probe CTD‐322006 used for FISH analysis. (E) STIM2 gene expression analysis . Relative expression of STIM2 blood mRNA in the proband (III‐2), his father (II‐2), and his sister (III‐1), compared to 10 healthy controls. STIM2 showed a significant overexpression in III‐2, while a normal expression level was found in II‐2 and III‐1 compared to controls. Data were normalized against TBP as housekeeping gene; similar results were obtained using GAPDH as normalizer (data not shown). [Corrections added on 21 May 2020 after first publication: Figure 2 has been corrected.]

Article Snippet: All assays were provided by Thermo Fisher Scientific (TaqMan Gene Expression Assays: ID# Hs00372705_m1 STIM2; Hs99999905_m1 GAPDH; Hs99999910_m1 TBP).

Techniques: Translocation Assay, Hybridization, Sequencing, Gene Expression, Expressing, Over Expression