Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: (A) Quantitative proteomics analysis of CMTM6-proficient (WT) and -deficient (KO) 8505C cells. Fold changes of significantly differentially expressed proteins (two-sided Student’s t test, p < 0.05) are depicted. (B) List of top 10 downregulated proteins in CMTM6-deficient 8505C cells. (C) Western blot analysis of CMTM6, CD58, and PD-L1 expression in parental 8505C cells (WT) and independent CMTM6-knockout clonal cells (CMTM6 KO). HSP90 served as a control. (D) Schematic illustration of the flow cytometry-based haploid genetic screen for modulators of CD58 expression. (E) Identification of modulators of CD58 expression by the haploid genetic screen depicted in (D). Each dot represents an individual gene, with the x axis indicating the number of disruptive insertions in each gene, and the y axis showing the fold changes in the frequency of unique insertions in the CD58 high population compared to the CD58 low population. Genes with a significant enrichment of insertions (two-sided Fisher’s exact test, FDR-corrected p < 0.05) in either the CD58 high or CD58 low populations are represented by light orange and blue dots, respectively. (F and G) CD58 expression levels in parental HAP1 cells (WT) and independent CMTM6-knockout clonal cells (CMTM6 KO). Levels of CD58 expression were determined by flow cytometry (F) and CMTM6 expression was analyzed by Western blot (G). HSP90 served as a control in the Western blot analysis. (H and I) Flow cytometry analysis of CD58 and PD-L1 expression in wild-type (WT), CMTM6-knockout (CMTM6 KO), CMTM6-overexpressing (WT + CMTM6 OE), and CMTM6-reconstituted (CMTM6 KO + CMTM6 OE) 8505C (H) and A375 cells with or without IFNγ exposure (I). Data represent the mean ± standard deviation of triplicates and were analyzed using a two-way ANOVA test (with Tukey’s multiple comparisons test). A p value greater than 0.05 indicates non-significance (ns), while a p value less than 0.0001 is denoted as **.

Article Snippet: A375 and RKO cells were obtained from the American Type Culture Collection (ATCC), while 8505C, Ramos, OCI-AML2, and BJAB cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).

Techniques: Quantitative Proteomics, Western Blot, Expressing, Knock-Out, Control, Flow Cytometry, Standard Deviation

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: (A) qPCR analysis of mRNA levels of CD58 in CMTM6-deficient and -proficient A375, 8505C, and RKO cells. (B) Stability of cell surface-expressed CD58, PD-L1, and MHC class I in parental (WT) and CMTM6-deficient (CMTM6 KO#6, CMTM6 KO#12) A375 cells. A375 cells were treated with IFNγ for 24 h and then individually incubated with APC-conjugated antibodies specific for CD58, PD-L1, or MHC class I at 4°C. After removing unbound antibodies, the cells were further incubated at 37°C for the indicated time periods, and the APC signal was measured by flow cytometry. The percentage of signal remaining at the indicated time points relative to time 0 is shown. (C) Stability of cell surface-expressed CD58 in the parental (WT) and CMTM6-deficient (CMTM6 KO#6, CMTM6 KO#12) A375 cells in the presence of the proteasome inhibitor bortezomib (Bor.) or the lysosome inhibitor concanamycin A (ConA). The untreated samples presented in (B) served as the control. Data acquisition and presentation were performed as described in (B). (D) Western blot analysis of cell lysates and indicated immunoprecipitates from A375 cells. HSP90 served as a control. (E) Western blot analysis of cell lysates and indicated immunoprecipitates by cell surface immunoprecipitation from A375 cells. For the cell surface immunoprecipitation, live cells were incubated with antibodies that recognize the extracellular domains of CD58 or PD-L1. After removal of unbound antibodies, the cells were lysed for (co)immunoprecipitation. HSP90 served as a control. The triangles indicate the position of background bands, which are present when the anti-CD58 antibody (R&D Cat# AF1689) was used for detection, whereas the anti-CD58 antibody (BioLegend, Cat# 330924) does not produce such background signal. Data represent mean ± standard deviation of at least triplicates (A–C) and were analyzed using unpaired Student’s t test. Statistical significance is indicated by *p < 0.05.

Article Snippet: A375 and RKO cells were obtained from the American Type Culture Collection (ATCC), while 8505C, Ramos, OCI-AML2, and BJAB cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).

Techniques: Incubation, Flow Cytometry, Control, Western Blot, Immunoprecipitation, Standard Deviation

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A375 and RKO cells were obtained from the American Type Culture Collection (ATCC), while 8505C, Ramos, OCI-AML2, and BJAB cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).

Techniques: Generated, Conjugation Assay, Control, Virus, Recombinant, Mutagenesis, cDNA Synthesis, Staining, Plasmid Preparation, Sequencing, CRISPR, Knock-Out, shRNA, Software

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Neutrophils express membrane Orai1. Isolated mouse and human PMNs, or 2-day differentiated HL-60 were assessed for RNA, DNA, or protein expression. (A) Protein expression levels were measured from the average mean fluorescence intensity (MFI) of paired flow cytometry samples of HL-60 cells transfected with negative control or Orai1-specific siRNA (n = 10) and PMNs (n = 4). Background fluorescence resulting from nonspecific antibody binding was measured by analyzing cells treated with a blocking decoy peptide against which the Orai1 antibody was originally raised. (B) Relative expression levels of Orai1 mRNA compared with RPL mRNA were measured for siRNA-transfected, control siRNA-transfected, or mock-transfected HL-60 cells by quantitative PCR. Data expressed as Orail mRNA copy number relative to RPL after PCR amplification. (C) Relative genomic DNA expression of Orai1 in WT versus Orai1−/+ mice measured by electrophoresis of amplified DNA from mouse tissues. Bars represent average densitometry measurements from 18 Orai1−/+ and 6 littermate WT mice ± SE. (D) Orai1 protein expression measured from the average MFI of paired cytometric measurements in WT and Orai1+/− mouse PMNs (n = 3). *P < .05; ***P < .001.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Isolation, Expressing, Fluorescence, Flow Cytometry, Transfection, Negative Control, Binding Assay, Blocking Assay, Real-time Polymerase Chain Reaction, Amplification, Electrophoresis

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Calcium dynamics via Orai1 supports GPCR-mediated PMN recruitment. PMNs isolated from murine Orai1+/+ (WT), or Orai1−/+ littermates, 2-day differentiated HL-60 cells, or freshly isolated human PMNs were loaded with fura-2 AM, perfused at a shear stress of 2 dyne/cm2 into a microfluidic flow chamber consisting of an L-cell monolayer substrate expressing E-selectin, and then 0.1μM fMLP was perfused just after time 0 and ratio-imaged by fluorescence microscopy. (A) Representative images of a human PMN decelerating, arresting, and undergoing shape polarization show fura-2 emission, with the intensity of red representing 340 nm excitation (calcium bound) and green representing 380nM excitation (calcium unbound). The average calcium concentration is shown in 1-second increments, and the percentage of cells arrested is shown in 10-second increments. (B) Percentage of arrested and polarized in WT and Orai1−/+ PMNs is based on analysis of more than 150 cells from 4 paired experiments. WT cells had significantly higher arrest and polarization than Orai1−/+ at time points beyond 30 seconds. **P < .01. (C) HL-60 were transfected with negative control, or Orai1-specific siRNA as indicated, were differentiated for 2 days with 1.3% DMSO and were imaged in the flow channels responding to 0.1μM fMLP as in panel A (n = 10 separate experiments). Negative control transfectants were treated with 2-APB or U73122 to completely block SOCE or store release. For Ca2+ quantified at 60 seconds. *P < .05. **P < .01. (D) Human PMNs were observed in absence of inhibition (n = 6), in the presence of 100μM 2-APB (n = 4), or in the presence of 1μM U73122 (n = 4) as indicated. Cells treated with 2-APB had significantly lower arrest fraction at 30 seconds (P = .003) and significantly lower polarization between 50 seconds and 100 seconds (P values range from .007 to .045 compared with the controls). *P < .05; #P < .05 (1-tailed t test, P = .059 by 2-tailed t test).

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Isolation, Expressing, Fluorescence, Microscopy, Concentration Assay, Transfection, Negative Control, Blocking Assay, Inhibition

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Calcium influx through SOCE and Orai1 facilitates neutrophil arrest. (A) PMNs isolated from murine Orai1−/+, WT littermates (ie, Orai1+/+), 2-day differentiated HL-60 cells, or freshly isolated human PMNs were loaded with fura-2, incubated with 1 μM thapsigargin as indicated, and then perfused over a monolayer of L cells expressing E-selectin and ICAM-1 in the presence of 0.1mM ethyleneglycoltetraacetic acid. At time 0, 1.5mM CaCl2 was injected into the inlet reservoir triggering selectin-dependent rolling and calcium influx through store-operated calcium channels. (A) Panels indicate intracellular calcium concentration and arrested fraction from WT or Orai1−/+ PMNs as indicated. (B) HL-60 cells were transfected with scrambled control or specific Orai1 siRNA as indicated (both n = 10). Orai1 siRNA-transfected cells had significantly higher rolling fractions at 150 seconds, 160 seconds, and 170 seconds (P = .03, .01, and .03, respectively) than control transfected cells. (C) Human PMNs were analyzed in the presence of vehicle control (n = 6), 100μM 2-APB (n = 4), or 1μM U73122 (n = 4) as a function of time. Cells treated with 2-APB had significantly lower arrest fraction than control at 60 seconds (P = .01). All statistical comparisons in this figure are by 2-tailed t test: *P < .05; **P < .01.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Isolation, Incubation, Expressing, Injection, Concentration Assay, Transfection

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Intracellular calcium rises in synchrony with PMN arrest and polarization. (A) Data for human PMNs responding to fMLP stimulation are compiled from more than 200 individual observations onto 1 plot to compare the dynamics of calcium, arrest, and polarization. Arrest is represented here as the fraction of cells remaining rolling (ie, the inverse of arrest). (B) Data for human PMNs treated with thapsigargin and stimulated by the addition of 1.5mM CaCl2 are also compiled onto 1 plot to compare the dynamics of calcium arrest and polarization. Arrest is represented as in panel A. (C) Human and mouse PMNs were loaded with fluo-5f, perfused over a glass coverslip coated with recombinant E-selectin-Fc and ICAM-1-Fc, and imaged by TIRF microscopy. Representative images from 3 separate experiments are indicated. (D) Calcium (fluo-5F) intensity over 60 seconds, untreated PMNs had significantly higher relative fluo-5f signal than 2-APB treated cells at 15 seconds and higher time points (P < .01). (E) Calcium (fluo-5F) signal in mouse WT versus Orai1−/+ PMNs over 60 seconds. **P < .01. ***P < .001. Error bars represent the SE for each measured time point.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Recombinant, Microscopy

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Conceptual model of calcium regulation and integrin engagement during PMN recruitment in shear flow. (A) During rolling, release of calcium stores initiated by GPCR and PLC-β signals IP3-sensitive calciosomes that cooperate with Orai1 SOCE eliciting an upshift in β2-integrin activation via Rap-1 that is sensitive to the rapid and local release of calcium stores resulting in PMN arrest. (B) As the PMN transitions to arrest, integrin engagement under stress stabilizes a high affinity conformation, which triggers activation of PLC-γ. The additional calcium release caused by integrin engagement sustains SOCE-mediated calcium influx and reinforces signaling for strengthening of arrest. (C) Calcium signaling causes rearrangement of the actin cytoskeleton and activation of secondary messengers (PKC) necessary for shape polarization. Electrical depolarization of the membrane and refilling of calcium stores through SERCA pumps slowly diminish intracellular calcium.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Activation Assay

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