rabbit polyclonal anti tpcn2  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti tpcn2
    Knockdown of <t>TPCN2</t> induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P
    Rabbit Polyclonal Anti Tpcn2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti tpcn2/product/Alomone Labs
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti tpcn2 - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle"

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    Journal: Chinese Medical Journal

    doi: 10.1097/CM9.0000000000001893

    Knockdown of TPCN2 induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P
    Figure Legend Snippet: Knockdown of TPCN2 induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P

    Techniques Used: Flow Cytometry

    TPCN2 -knockdown inhibits the proliferation in SLE. (A) Expression level of TPCN2 in PBMCs of SLE cases ( n = 6) and healthy controls ( n = 7). ∗ P
    Figure Legend Snippet: TPCN2 -knockdown inhibits the proliferation in SLE. (A) Expression level of TPCN2 in PBMCs of SLE cases ( n = 6) and healthy controls ( n = 7). ∗ P

    Techniques Used: Expressing

    RNA-seq analyses of the effect of TPCN2 -knockdown on the gene expression profile. (A) The heatmap shows differential expression genes in sh#1 and sh#2 (in comparison with NC group). (B) The differential statistics of DEGs in NC groups vs . sh#1/sh#2. Blue columnar represent up-regulated DEGs. Red column represents down-regulated DEGs. (C) GO classification analysis of DEGs. NC group vs . sh#2. Molecular function – blue; Cellular components – red; Biological process – green. (D) Top 20 enriched KEGG pathways after silencing TPCN2 in Jurkat. The x-axis is the enrichment score, and the y-axis is the enriched pathways. DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; NC: Normal control; RNA-seq: RNA sequencing; Sh#1: shTPCN2#1; Sh#2: shTPCN2#2; TPCN2 : Two-pore segment channel 2.
    Figure Legend Snippet: RNA-seq analyses of the effect of TPCN2 -knockdown on the gene expression profile. (A) The heatmap shows differential expression genes in sh#1 and sh#2 (in comparison with NC group). (B) The differential statistics of DEGs in NC groups vs . sh#1/sh#2. Blue columnar represent up-regulated DEGs. Red column represents down-regulated DEGs. (C) GO classification analysis of DEGs. NC group vs . sh#2. Molecular function – blue; Cellular components – red; Biological process – green. (D) Top 20 enriched KEGG pathways after silencing TPCN2 in Jurkat. The x-axis is the enrichment score, and the y-axis is the enriched pathways. DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; NC: Normal control; RNA-seq: RNA sequencing; Sh#1: shTPCN2#1; Sh#2: shTPCN2#2; TPCN2 : Two-pore segment channel 2.

    Techniques Used: RNA Sequencing Assay, Expressing

    Representative enriched pathways in high-risk shTPCN2#2 through GSEA analysis. GSEA results showed that the G2/M checkpoint (A), inflammatory response (B), complement (C), and PI3K-AKT-mTOR (D) pathways were enriched in the sh#2 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. (E) The mRNA expression of some DEGs in Jurkat cells were detected by qRT-PCR. The RNA was extracted from cells knocked down of TPCN2 with two independent shRNA. The results were shown as the mean ± SD from three independent experiments. ∗ P
    Figure Legend Snippet: Representative enriched pathways in high-risk shTPCN2#2 through GSEA analysis. GSEA results showed that the G2/M checkpoint (A), inflammatory response (B), complement (C), and PI3K-AKT-mTOR (D) pathways were enriched in the sh#2 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. (E) The mRNA expression of some DEGs in Jurkat cells were detected by qRT-PCR. The RNA was extracted from cells knocked down of TPCN2 with two independent shRNA. The results were shown as the mean ± SD from three independent experiments. ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR, shRNA

    2) Product Images from "Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)"

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    Journal: bioRxiv

    doi: 10.1101/2021.09.28.462104

    Targeting TPC2 activity affects histamine-evoked P-selectin cell surface presentation in HUVEC. (A) Representative images of cell-surface P-selectin pools of control cells and cells treated with either trans-Ned 19 (10 µM) or TPC2-A1-N (10 µM). Dashed lines outline cells of interest. Bars, 20 µm. (B) Quantitative analysis of P-selectin surface signals confirmed the altered cell surface levels of P-selectin. Data represent mean ± SEM of at least 22 cells per condition from three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, ***p
    Figure Legend Snippet: Targeting TPC2 activity affects histamine-evoked P-selectin cell surface presentation in HUVEC. (A) Representative images of cell-surface P-selectin pools of control cells and cells treated with either trans-Ned 19 (10 µM) or TPC2-A1-N (10 µM). Dashed lines outline cells of interest. Bars, 20 µm. (B) Quantitative analysis of P-selectin surface signals confirmed the altered cell surface levels of P-selectin. Data represent mean ± SEM of at least 22 cells per condition from three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, ***p

    Techniques Used: Activity Assay

    Endothelial TPC2 inhibition impairs histamine-evoked leukocyte recruitment. Adhesion of primary human neutrophils to unstimulated (basal), histamine-activated or anti-P-selectin-antibody-blocked HUVEC monolayers under flow. (A) Representative images after histamine treatment of control (top) and trans-Ned19-treated HUVEC monolayers (bottom). Arrows mark firmly attached leukocytes. Scale bars, 100 μm. Analysis of (B) PMN adhesion, (C) rolling, and (D) rolling velocities on control or trans-Ned 19-treated HUVEC monolayers. Data represent means ± SEM from five independent experiments (n=5). Data was statistically analyzed by one way ANOVA (adherent cells), Kruskal-Wallis test (rolling cells) and Mann-Whitney test (rolling velocity) (*p
    Figure Legend Snippet: Endothelial TPC2 inhibition impairs histamine-evoked leukocyte recruitment. Adhesion of primary human neutrophils to unstimulated (basal), histamine-activated or anti-P-selectin-antibody-blocked HUVEC monolayers under flow. (A) Representative images after histamine treatment of control (top) and trans-Ned19-treated HUVEC monolayers (bottom). Arrows mark firmly attached leukocytes. Scale bars, 100 μm. Analysis of (B) PMN adhesion, (C) rolling, and (D) rolling velocities on control or trans-Ned 19-treated HUVEC monolayers. Data represent means ± SEM from five independent experiments (n=5). Data was statistically analyzed by one way ANOVA (adherent cells), Kruskal-Wallis test (rolling cells) and Mann-Whitney test (rolling velocity) (*p

    Techniques Used: Inhibition, MANN-WHITNEY

    Targeting TPC2 affects endolysosomal cholesterol balance. Cells were either solvent-treated (Ctrl) or treated with the TPC-targeting compounds TPC1-A1-N (10 µM) and trans-Ned 19 (10 µM). U18666A (U18, 2µg/mL), the direct inhibitor of the endolysosomal cholesterol transporter NPC1, served as an internal reference. Filipin was used to detect cholesterol, CD63 or Lysotracker were used to identify endolysosomes. (A) Representative confocal images of treatment-induced endolysosomal cholesterol contents. Scale bars, 20 µm. To better visualize the endolysosomal cholesterol accumulation, filipin signal intensities were color-encoded (see calibration bar). (B) Representative confocal images of treatment-induced endolysosomal CHIM-L-AF488 contents. Scale bars, 20 µm. (C) The amount of endolysosomal cholesterol was assessed by calculating the Manders’ colocalization coefficients M1 of CD63 and filipin signals from z-stacks of individual cells. (D) The amount of endolysosomal CHIM-L-AF488 was assessed by calculating the Manders’ colocalization coefficients M1 of Lysotracker and CHIM-L-AF488 signals from z-stacks of individual cells. Data are presented as mean ± SEM of at least 41 cells from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s post-test (n.s., not significant, *p
    Figure Legend Snippet: Targeting TPC2 affects endolysosomal cholesterol balance. Cells were either solvent-treated (Ctrl) or treated with the TPC-targeting compounds TPC1-A1-N (10 µM) and trans-Ned 19 (10 µM). U18666A (U18, 2µg/mL), the direct inhibitor of the endolysosomal cholesterol transporter NPC1, served as an internal reference. Filipin was used to detect cholesterol, CD63 or Lysotracker were used to identify endolysosomes. (A) Representative confocal images of treatment-induced endolysosomal cholesterol contents. Scale bars, 20 µm. To better visualize the endolysosomal cholesterol accumulation, filipin signal intensities were color-encoded (see calibration bar). (B) Representative confocal images of treatment-induced endolysosomal CHIM-L-AF488 contents. Scale bars, 20 µm. (C) The amount of endolysosomal cholesterol was assessed by calculating the Manders’ colocalization coefficients M1 of CD63 and filipin signals from z-stacks of individual cells. (D) The amount of endolysosomal CHIM-L-AF488 was assessed by calculating the Manders’ colocalization coefficients M1 of Lysotracker and CHIM-L-AF488 signals from z-stacks of individual cells. Data are presented as mean ± SEM of at least 41 cells from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s post-test (n.s., not significant, *p

    Techniques Used:

    TPC2 inhibition impairs endolysosomal Ca 2+ release. Ratiometric in vivo Ca 2+ measurements showing a decreased Ca 2+ release from endolysosomal stores of cells treated with the TPC2 inhibitor trans-Ned 19 (10 µM). Blocking lysosomal Ca 2+ reuptake with the VATPase inhibitor bafilomycin A1 (250 nM) was used to amplify Ca 2+ signals. (A) Representative ratiometric time course measurement of endolysosomal Ca 2+ release upon bafilomycin A1 stimulation of control cells and trans-Ned19 treated cells. (B) Quantification of Ca 2+ release of 24 cells per condition from four independent experiments. Ca 2+ response is depicted in relation to the control. Two-tailed Student’s t-test (*p
    Figure Legend Snippet: TPC2 inhibition impairs endolysosomal Ca 2+ release. Ratiometric in vivo Ca 2+ measurements showing a decreased Ca 2+ release from endolysosomal stores of cells treated with the TPC2 inhibitor trans-Ned 19 (10 µM). Blocking lysosomal Ca 2+ reuptake with the VATPase inhibitor bafilomycin A1 (250 nM) was used to amplify Ca 2+ signals. (A) Representative ratiometric time course measurement of endolysosomal Ca 2+ release upon bafilomycin A1 stimulation of control cells and trans-Ned19 treated cells. (B) Quantification of Ca 2+ release of 24 cells per condition from four independent experiments. Ca 2+ response is depicted in relation to the control. Two-tailed Student’s t-test (*p

    Techniques Used: Inhibition, In Vivo, Blocking Assay, Two Tailed Test

    TPC2-specific activation with TPC2-A1-N as determined via a TPC2-based ratiometric Ca 2+ indicator. Representative confocal images of wild-type (A) and pore-dead (B) TPC2-GCaMP-mApple constructs expressed in HUVEC. GCaMP fluorescence signal is elicited upon stimulation with the TPC2 activator TPC2-A1-N and signal intensity is depicted as a heatmap. (C) Comparison of representative time-resolved fluorescence ratios in TPC2-A1-N stimulated cells expressing either the active TPC2 (black trace) or the pore-dead TPC2 (grey trace). Note that ionomycin-induced unspecific elevation of cytosolic Ca 2+ levels was still observable in both cases.
    Figure Legend Snippet: TPC2-specific activation with TPC2-A1-N as determined via a TPC2-based ratiometric Ca 2+ indicator. Representative confocal images of wild-type (A) and pore-dead (B) TPC2-GCaMP-mApple constructs expressed in HUVEC. GCaMP fluorescence signal is elicited upon stimulation with the TPC2 activator TPC2-A1-N and signal intensity is depicted as a heatmap. (C) Comparison of representative time-resolved fluorescence ratios in TPC2-A1-N stimulated cells expressing either the active TPC2 (black trace) or the pore-dead TPC2 (grey trace). Note that ionomycin-induced unspecific elevation of cytosolic Ca 2+ levels was still observable in both cases.

    Techniques Used: Activation Assay, Construct, Fluorescence, Expressing

    TPC2 is required for efficient CD63 transport to WPB but does not affect WPB exocytosis. (A) CD63 LEL to WPB transport was followed in cells treated with the TPC-targeting compounds (upper panel) or transfected with either control siRNA or TPC1/2 siRNA (lower panel). WPB were detected via VWF staining (green) and were analyzed for the amount of transferred anti-CD63 antibodies (red). Nuclei were visualized by DAPI (grey). Boxed areas indicate the regions magnified in the insets. Scale bars, 20 µm. To quantify the amount of anti-CD63 antibodies on WPB, Manders’ colocalization coefficients (MCC) were calculated from z-stack images of individual cells. Data represent mean ± SEM of at least 45 cells of three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, *p
    Figure Legend Snippet: TPC2 is required for efficient CD63 transport to WPB but does not affect WPB exocytosis. (A) CD63 LEL to WPB transport was followed in cells treated with the TPC-targeting compounds (upper panel) or transfected with either control siRNA or TPC1/2 siRNA (lower panel). WPB were detected via VWF staining (green) and were analyzed for the amount of transferred anti-CD63 antibodies (red). Nuclei were visualized by DAPI (grey). Boxed areas indicate the regions magnified in the insets. Scale bars, 20 µm. To quantify the amount of anti-CD63 antibodies on WPB, Manders’ colocalization coefficients (MCC) were calculated from z-stack images of individual cells. Data represent mean ± SEM of at least 45 cells of three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, *p

    Techniques Used: Transfection, Staining

    3) Product Images from "The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction"

    Article Title: The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction

    Journal: Cells

    doi: 10.3390/cells8101144

    Decrease in [Ca 2+ ] i rise in response to NE in SMCs transfected with siRNA against TPC1. ( A ) Kinetics of [Ca 2+ ] i rise in SMCs transfected with siRNA against TPC1 and nontarget siRNA. The curves from one of four transfection experiments are presented. Each point at the curves is an average of six parallel measurements. ( B ) Calcium responses to NE (100 μM) and angiotensin II (0.1 μM) in SMCs transfected with nontarget siRNA, and siRNA against TPC1 and against TPC2. [Ca 2+ ] i rise in SMCs transfected with nontarget siRNA is taken as 100%. The mean values + SEM from four independent transfection experiments with different SMCs preparations are presented. There is significant difference between the responses of SMCs transfected with anti-TPC1 siRNA and with nontarget (** p
    Figure Legend Snippet: Decrease in [Ca 2+ ] i rise in response to NE in SMCs transfected with siRNA against TPC1. ( A ) Kinetics of [Ca 2+ ] i rise in SMCs transfected with siRNA against TPC1 and nontarget siRNA. The curves from one of four transfection experiments are presented. Each point at the curves is an average of six parallel measurements. ( B ) Calcium responses to NE (100 μM) and angiotensin II (0.1 μM) in SMCs transfected with nontarget siRNA, and siRNA against TPC1 and against TPC2. [Ca 2+ ] i rise in SMCs transfected with nontarget siRNA is taken as 100%. The mean values + SEM from four independent transfection experiments with different SMCs preparations are presented. There is significant difference between the responses of SMCs transfected with anti-TPC1 siRNA and with nontarget (** p

    Techniques Used: Transfection

    Levels of TPC1 and TPC2 mRNA relative to β-actin mRNA ( A ). Western-blot of TPC1 and TPC2 proteins ( B ) isolated from rat aorta SMCs and relative levels of TPC1 and TPC2 ( C ). The values panel A are the means of four measurements in different SMCs preparation + SEM (* p
    Figure Legend Snippet: Levels of TPC1 and TPC2 mRNA relative to β-actin mRNA ( A ). Western-blot of TPC1 and TPC2 proteins ( B ) isolated from rat aorta SMCs and relative levels of TPC1 and TPC2 ( C ). The values panel A are the means of four measurements in different SMCs preparation + SEM (* p

    Techniques Used: Western Blot, Isolation

    4) Product Images from "The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction"

    Article Title: The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction

    Journal: Cells

    doi: 10.3390/cells8101144

    Decrease in [Ca 2+ ] i rise in response to NE in SMCs transfected with siRNA against TPC1. ( A ) Kinetics of [Ca 2+ ] i rise in SMCs transfected with siRNA against TPC1 and nontarget siRNA. The curves from one of four transfection experiments are presented. Each point at the curves is an average of six parallel measurements. ( B ) Calcium responses to NE (100 μM) and angiotensin II (0.1 μM) in SMCs transfected with nontarget siRNA, and siRNA against TPC1 and against TPC2. [Ca 2+ ] i rise in SMCs transfected with nontarget siRNA is taken as 100%. The mean values + SEM from four independent transfection experiments with different SMCs preparations are presented. There is significant difference between the responses of SMCs transfected with anti-TPC1 siRNA and with nontarget (** p
    Figure Legend Snippet: Decrease in [Ca 2+ ] i rise in response to NE in SMCs transfected with siRNA against TPC1. ( A ) Kinetics of [Ca 2+ ] i rise in SMCs transfected with siRNA against TPC1 and nontarget siRNA. The curves from one of four transfection experiments are presented. Each point at the curves is an average of six parallel measurements. ( B ) Calcium responses to NE (100 μM) and angiotensin II (0.1 μM) in SMCs transfected with nontarget siRNA, and siRNA against TPC1 and against TPC2. [Ca 2+ ] i rise in SMCs transfected with nontarget siRNA is taken as 100%. The mean values + SEM from four independent transfection experiments with different SMCs preparations are presented. There is significant difference between the responses of SMCs transfected with anti-TPC1 siRNA and with nontarget (** p

    Techniques Used: Transfection

    Levels of TPC1 and TPC2 mRNA relative to β-actin mRNA ( A ). Western-blot of TPC1 and TPC2 proteins ( B ) isolated from rat aorta SMCs and relative levels of TPC1 and TPC2 ( C ). The values panel A are the means of four measurements in different SMCs preparation + SEM (* p
    Figure Legend Snippet: Levels of TPC1 and TPC2 mRNA relative to β-actin mRNA ( A ). Western-blot of TPC1 and TPC2 proteins ( B ) isolated from rat aorta SMCs and relative levels of TPC1 and TPC2 ( C ). The values panel A are the means of four measurements in different SMCs preparation + SEM (* p

    Techniques Used: Western Blot, Isolation

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    Alomone Labs rabbit polyclonal anti tpcn2
    Knockdown of <t>TPCN2</t> induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P
    Rabbit Polyclonal Anti Tpcn2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti tpcn2/product/Alomone Labs
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti tpcn2 - by Bioz Stars, 2022-12
    94/100 stars
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    Knockdown of TPCN2 induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: Knockdown of TPCN2 induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: Flow Cytometry

    TPCN2 -knockdown inhibits the proliferation in SLE. (A) Expression level of TPCN2 in PBMCs of SLE cases ( n = 6) and healthy controls ( n = 7). ∗ P

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: TPCN2 -knockdown inhibits the proliferation in SLE. (A) Expression level of TPCN2 in PBMCs of SLE cases ( n = 6) and healthy controls ( n = 7). ∗ P

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: Expressing

    RNA-seq analyses of the effect of TPCN2 -knockdown on the gene expression profile. (A) The heatmap shows differential expression genes in sh#1 and sh#2 (in comparison with NC group). (B) The differential statistics of DEGs in NC groups vs . sh#1/sh#2. Blue columnar represent up-regulated DEGs. Red column represents down-regulated DEGs. (C) GO classification analysis of DEGs. NC group vs . sh#2. Molecular function – blue; Cellular components – red; Biological process – green. (D) Top 20 enriched KEGG pathways after silencing TPCN2 in Jurkat. The x-axis is the enrichment score, and the y-axis is the enriched pathways. DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; NC: Normal control; RNA-seq: RNA sequencing; Sh#1: shTPCN2#1; Sh#2: shTPCN2#2; TPCN2 : Two-pore segment channel 2.

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: RNA-seq analyses of the effect of TPCN2 -knockdown on the gene expression profile. (A) The heatmap shows differential expression genes in sh#1 and sh#2 (in comparison with NC group). (B) The differential statistics of DEGs in NC groups vs . sh#1/sh#2. Blue columnar represent up-regulated DEGs. Red column represents down-regulated DEGs. (C) GO classification analysis of DEGs. NC group vs . sh#2. Molecular function – blue; Cellular components – red; Biological process – green. (D) Top 20 enriched KEGG pathways after silencing TPCN2 in Jurkat. The x-axis is the enrichment score, and the y-axis is the enriched pathways. DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; NC: Normal control; RNA-seq: RNA sequencing; Sh#1: shTPCN2#1; Sh#2: shTPCN2#2; TPCN2 : Two-pore segment channel 2.

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: RNA Sequencing Assay, Expressing

    Representative enriched pathways in high-risk shTPCN2#2 through GSEA analysis. GSEA results showed that the G2/M checkpoint (A), inflammatory response (B), complement (C), and PI3K-AKT-mTOR (D) pathways were enriched in the sh#2 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. (E) The mRNA expression of some DEGs in Jurkat cells were detected by qRT-PCR. The RNA was extracted from cells knocked down of TPCN2 with two independent shRNA. The results were shown as the mean ± SD from three independent experiments. ∗ P

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: Representative enriched pathways in high-risk shTPCN2#2 through GSEA analysis. GSEA results showed that the G2/M checkpoint (A), inflammatory response (B), complement (C), and PI3K-AKT-mTOR (D) pathways were enriched in the sh#2 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. (E) The mRNA expression of some DEGs in Jurkat cells were detected by qRT-PCR. The RNA was extracted from cells knocked down of TPCN2 with two independent shRNA. The results were shown as the mean ± SD from three independent experiments. ∗ P

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: Expressing, Quantitative RT-PCR, shRNA

    Targeting TPC2 activity affects histamine-evoked P-selectin cell surface presentation in HUVEC. (A) Representative images of cell-surface P-selectin pools of control cells and cells treated with either trans-Ned 19 (10 µM) or TPC2-A1-N (10 µM). Dashed lines outline cells of interest. Bars, 20 µm. (B) Quantitative analysis of P-selectin surface signals confirmed the altered cell surface levels of P-selectin. Data represent mean ± SEM of at least 22 cells per condition from three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, ***p

    Journal: bioRxiv

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    doi: 10.1101/2021.09.28.462104

    Figure Lengend Snippet: Targeting TPC2 activity affects histamine-evoked P-selectin cell surface presentation in HUVEC. (A) Representative images of cell-surface P-selectin pools of control cells and cells treated with either trans-Ned 19 (10 µM) or TPC2-A1-N (10 µM). Dashed lines outline cells of interest. Bars, 20 µm. (B) Quantitative analysis of P-selectin surface signals confirmed the altered cell surface levels of P-selectin. Data represent mean ± SEM of at least 22 cells per condition from three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, ***p

    Article Snippet: Antibodies and labeling reagentsThe following primary antibodies were used: Mouse anti-CD63-TRITC (sc-5275, 1:100) and mouse anti-LAMP2 (sc-18822, 1:50) were obtained from Santa Cruz, rabbit anti-VWF (A0082, 1:1000) and rabbit anti-VWF-HRP (P0226, 1:8000) used for sandwich ELISA were purchased from Dako, sheep anti-VWF (ab11713, 1:200-400) and rabbit anti-TPC1 (ab94731, 1:200) were from Abcam, rabbit anti-TRPML1 was from Thermo Fisher Scientific (PA1-464741, 1:200), rabbit anti-TPC2 from Alomone Labs (ACC-072, 1:300), mouse anti-β-actin from Sigma (A5441, 1:5000) and sheep anti-P-selectin/CD62P from R+D Systems (AF137).

    Techniques: Activity Assay

    Endothelial TPC2 inhibition impairs histamine-evoked leukocyte recruitment. Adhesion of primary human neutrophils to unstimulated (basal), histamine-activated or anti-P-selectin-antibody-blocked HUVEC monolayers under flow. (A) Representative images after histamine treatment of control (top) and trans-Ned19-treated HUVEC monolayers (bottom). Arrows mark firmly attached leukocytes. Scale bars, 100 μm. Analysis of (B) PMN adhesion, (C) rolling, and (D) rolling velocities on control or trans-Ned 19-treated HUVEC monolayers. Data represent means ± SEM from five independent experiments (n=5). Data was statistically analyzed by one way ANOVA (adherent cells), Kruskal-Wallis test (rolling cells) and Mann-Whitney test (rolling velocity) (*p

    Journal: bioRxiv

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    doi: 10.1101/2021.09.28.462104

    Figure Lengend Snippet: Endothelial TPC2 inhibition impairs histamine-evoked leukocyte recruitment. Adhesion of primary human neutrophils to unstimulated (basal), histamine-activated or anti-P-selectin-antibody-blocked HUVEC monolayers under flow. (A) Representative images after histamine treatment of control (top) and trans-Ned19-treated HUVEC monolayers (bottom). Arrows mark firmly attached leukocytes. Scale bars, 100 μm. Analysis of (B) PMN adhesion, (C) rolling, and (D) rolling velocities on control or trans-Ned 19-treated HUVEC monolayers. Data represent means ± SEM from five independent experiments (n=5). Data was statistically analyzed by one way ANOVA (adherent cells), Kruskal-Wallis test (rolling cells) and Mann-Whitney test (rolling velocity) (*p

    Article Snippet: Antibodies and labeling reagentsThe following primary antibodies were used: Mouse anti-CD63-TRITC (sc-5275, 1:100) and mouse anti-LAMP2 (sc-18822, 1:50) were obtained from Santa Cruz, rabbit anti-VWF (A0082, 1:1000) and rabbit anti-VWF-HRP (P0226, 1:8000) used for sandwich ELISA were purchased from Dako, sheep anti-VWF (ab11713, 1:200-400) and rabbit anti-TPC1 (ab94731, 1:200) were from Abcam, rabbit anti-TRPML1 was from Thermo Fisher Scientific (PA1-464741, 1:200), rabbit anti-TPC2 from Alomone Labs (ACC-072, 1:300), mouse anti-β-actin from Sigma (A5441, 1:5000) and sheep anti-P-selectin/CD62P from R+D Systems (AF137).

    Techniques: Inhibition, MANN-WHITNEY

    Targeting TPC2 affects endolysosomal cholesterol balance. Cells were either solvent-treated (Ctrl) or treated with the TPC-targeting compounds TPC1-A1-N (10 µM) and trans-Ned 19 (10 µM). U18666A (U18, 2µg/mL), the direct inhibitor of the endolysosomal cholesterol transporter NPC1, served as an internal reference. Filipin was used to detect cholesterol, CD63 or Lysotracker were used to identify endolysosomes. (A) Representative confocal images of treatment-induced endolysosomal cholesterol contents. Scale bars, 20 µm. To better visualize the endolysosomal cholesterol accumulation, filipin signal intensities were color-encoded (see calibration bar). (B) Representative confocal images of treatment-induced endolysosomal CHIM-L-AF488 contents. Scale bars, 20 µm. (C) The amount of endolysosomal cholesterol was assessed by calculating the Manders’ colocalization coefficients M1 of CD63 and filipin signals from z-stacks of individual cells. (D) The amount of endolysosomal CHIM-L-AF488 was assessed by calculating the Manders’ colocalization coefficients M1 of Lysotracker and CHIM-L-AF488 signals from z-stacks of individual cells. Data are presented as mean ± SEM of at least 41 cells from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s post-test (n.s., not significant, *p

    Journal: bioRxiv

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    doi: 10.1101/2021.09.28.462104

    Figure Lengend Snippet: Targeting TPC2 affects endolysosomal cholesterol balance. Cells were either solvent-treated (Ctrl) or treated with the TPC-targeting compounds TPC1-A1-N (10 µM) and trans-Ned 19 (10 µM). U18666A (U18, 2µg/mL), the direct inhibitor of the endolysosomal cholesterol transporter NPC1, served as an internal reference. Filipin was used to detect cholesterol, CD63 or Lysotracker were used to identify endolysosomes. (A) Representative confocal images of treatment-induced endolysosomal cholesterol contents. Scale bars, 20 µm. To better visualize the endolysosomal cholesterol accumulation, filipin signal intensities were color-encoded (see calibration bar). (B) Representative confocal images of treatment-induced endolysosomal CHIM-L-AF488 contents. Scale bars, 20 µm. (C) The amount of endolysosomal cholesterol was assessed by calculating the Manders’ colocalization coefficients M1 of CD63 and filipin signals from z-stacks of individual cells. (D) The amount of endolysosomal CHIM-L-AF488 was assessed by calculating the Manders’ colocalization coefficients M1 of Lysotracker and CHIM-L-AF488 signals from z-stacks of individual cells. Data are presented as mean ± SEM of at least 41 cells from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s post-test (n.s., not significant, *p

    Article Snippet: Antibodies and labeling reagentsThe following primary antibodies were used: Mouse anti-CD63-TRITC (sc-5275, 1:100) and mouse anti-LAMP2 (sc-18822, 1:50) were obtained from Santa Cruz, rabbit anti-VWF (A0082, 1:1000) and rabbit anti-VWF-HRP (P0226, 1:8000) used for sandwich ELISA were purchased from Dako, sheep anti-VWF (ab11713, 1:200-400) and rabbit anti-TPC1 (ab94731, 1:200) were from Abcam, rabbit anti-TRPML1 was from Thermo Fisher Scientific (PA1-464741, 1:200), rabbit anti-TPC2 from Alomone Labs (ACC-072, 1:300), mouse anti-β-actin from Sigma (A5441, 1:5000) and sheep anti-P-selectin/CD62P from R+D Systems (AF137).

    Techniques:

    TPC2 inhibition impairs endolysosomal Ca 2+ release. Ratiometric in vivo Ca 2+ measurements showing a decreased Ca 2+ release from endolysosomal stores of cells treated with the TPC2 inhibitor trans-Ned 19 (10 µM). Blocking lysosomal Ca 2+ reuptake with the VATPase inhibitor bafilomycin A1 (250 nM) was used to amplify Ca 2+ signals. (A) Representative ratiometric time course measurement of endolysosomal Ca 2+ release upon bafilomycin A1 stimulation of control cells and trans-Ned19 treated cells. (B) Quantification of Ca 2+ release of 24 cells per condition from four independent experiments. Ca 2+ response is depicted in relation to the control. Two-tailed Student’s t-test (*p

    Journal: bioRxiv

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    doi: 10.1101/2021.09.28.462104

    Figure Lengend Snippet: TPC2 inhibition impairs endolysosomal Ca 2+ release. Ratiometric in vivo Ca 2+ measurements showing a decreased Ca 2+ release from endolysosomal stores of cells treated with the TPC2 inhibitor trans-Ned 19 (10 µM). Blocking lysosomal Ca 2+ reuptake with the VATPase inhibitor bafilomycin A1 (250 nM) was used to amplify Ca 2+ signals. (A) Representative ratiometric time course measurement of endolysosomal Ca 2+ release upon bafilomycin A1 stimulation of control cells and trans-Ned19 treated cells. (B) Quantification of Ca 2+ release of 24 cells per condition from four independent experiments. Ca 2+ response is depicted in relation to the control. Two-tailed Student’s t-test (*p

    Article Snippet: Antibodies and labeling reagentsThe following primary antibodies were used: Mouse anti-CD63-TRITC (sc-5275, 1:100) and mouse anti-LAMP2 (sc-18822, 1:50) were obtained from Santa Cruz, rabbit anti-VWF (A0082, 1:1000) and rabbit anti-VWF-HRP (P0226, 1:8000) used for sandwich ELISA were purchased from Dako, sheep anti-VWF (ab11713, 1:200-400) and rabbit anti-TPC1 (ab94731, 1:200) were from Abcam, rabbit anti-TRPML1 was from Thermo Fisher Scientific (PA1-464741, 1:200), rabbit anti-TPC2 from Alomone Labs (ACC-072, 1:300), mouse anti-β-actin from Sigma (A5441, 1:5000) and sheep anti-P-selectin/CD62P from R+D Systems (AF137).

    Techniques: Inhibition, In Vivo, Blocking Assay, Two Tailed Test

    TPC2-specific activation with TPC2-A1-N as determined via a TPC2-based ratiometric Ca 2+ indicator. Representative confocal images of wild-type (A) and pore-dead (B) TPC2-GCaMP-mApple constructs expressed in HUVEC. GCaMP fluorescence signal is elicited upon stimulation with the TPC2 activator TPC2-A1-N and signal intensity is depicted as a heatmap. (C) Comparison of representative time-resolved fluorescence ratios in TPC2-A1-N stimulated cells expressing either the active TPC2 (black trace) or the pore-dead TPC2 (grey trace). Note that ionomycin-induced unspecific elevation of cytosolic Ca 2+ levels was still observable in both cases.

    Journal: bioRxiv

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    doi: 10.1101/2021.09.28.462104

    Figure Lengend Snippet: TPC2-specific activation with TPC2-A1-N as determined via a TPC2-based ratiometric Ca 2+ indicator. Representative confocal images of wild-type (A) and pore-dead (B) TPC2-GCaMP-mApple constructs expressed in HUVEC. GCaMP fluorescence signal is elicited upon stimulation with the TPC2 activator TPC2-A1-N and signal intensity is depicted as a heatmap. (C) Comparison of representative time-resolved fluorescence ratios in TPC2-A1-N stimulated cells expressing either the active TPC2 (black trace) or the pore-dead TPC2 (grey trace). Note that ionomycin-induced unspecific elevation of cytosolic Ca 2+ levels was still observable in both cases.

    Article Snippet: Antibodies and labeling reagentsThe following primary antibodies were used: Mouse anti-CD63-TRITC (sc-5275, 1:100) and mouse anti-LAMP2 (sc-18822, 1:50) were obtained from Santa Cruz, rabbit anti-VWF (A0082, 1:1000) and rabbit anti-VWF-HRP (P0226, 1:8000) used for sandwich ELISA were purchased from Dako, sheep anti-VWF (ab11713, 1:200-400) and rabbit anti-TPC1 (ab94731, 1:200) were from Abcam, rabbit anti-TRPML1 was from Thermo Fisher Scientific (PA1-464741, 1:200), rabbit anti-TPC2 from Alomone Labs (ACC-072, 1:300), mouse anti-β-actin from Sigma (A5441, 1:5000) and sheep anti-P-selectin/CD62P from R+D Systems (AF137).

    Techniques: Activation Assay, Construct, Fluorescence, Expressing

    TPC2 is required for efficient CD63 transport to WPB but does not affect WPB exocytosis. (A) CD63 LEL to WPB transport was followed in cells treated with the TPC-targeting compounds (upper panel) or transfected with either control siRNA or TPC1/2 siRNA (lower panel). WPB were detected via VWF staining (green) and were analyzed for the amount of transferred anti-CD63 antibodies (red). Nuclei were visualized by DAPI (grey). Boxed areas indicate the regions magnified in the insets. Scale bars, 20 µm. To quantify the amount of anti-CD63 antibodies on WPB, Manders’ colocalization coefficients (MCC) were calculated from z-stack images of individual cells. Data represent mean ± SEM of at least 45 cells of three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, *p

    Journal: bioRxiv

    Article Title: Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

    doi: 10.1101/2021.09.28.462104

    Figure Lengend Snippet: TPC2 is required for efficient CD63 transport to WPB but does not affect WPB exocytosis. (A) CD63 LEL to WPB transport was followed in cells treated with the TPC-targeting compounds (upper panel) or transfected with either control siRNA or TPC1/2 siRNA (lower panel). WPB were detected via VWF staining (green) and were analyzed for the amount of transferred anti-CD63 antibodies (red). Nuclei were visualized by DAPI (grey). Boxed areas indicate the regions magnified in the insets. Scale bars, 20 µm. To quantify the amount of anti-CD63 antibodies on WPB, Manders’ colocalization coefficients (MCC) were calculated from z-stack images of individual cells. Data represent mean ± SEM of at least 45 cells of three independent experiments and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n.s., not significant, *p

    Article Snippet: Antibodies and labeling reagentsThe following primary antibodies were used: Mouse anti-CD63-TRITC (sc-5275, 1:100) and mouse anti-LAMP2 (sc-18822, 1:50) were obtained from Santa Cruz, rabbit anti-VWF (A0082, 1:1000) and rabbit anti-VWF-HRP (P0226, 1:8000) used for sandwich ELISA were purchased from Dako, sheep anti-VWF (ab11713, 1:200-400) and rabbit anti-TPC1 (ab94731, 1:200) were from Abcam, rabbit anti-TRPML1 was from Thermo Fisher Scientific (PA1-464741, 1:200), rabbit anti-TPC2 from Alomone Labs (ACC-072, 1:300), mouse anti-β-actin from Sigma (A5441, 1:5000) and sheep anti-P-selectin/CD62P from R+D Systems (AF137).

    Techniques: Transfection, Staining

    Decrease in [Ca 2+ ] i rise in response to NE in SMCs transfected with siRNA against TPC1. ( A ) Kinetics of [Ca 2+ ] i rise in SMCs transfected with siRNA against TPC1 and nontarget siRNA. The curves from one of four transfection experiments are presented. Each point at the curves is an average of six parallel measurements. ( B ) Calcium responses to NE (100 μM) and angiotensin II (0.1 μM) in SMCs transfected with nontarget siRNA, and siRNA against TPC1 and against TPC2. [Ca 2+ ] i rise in SMCs transfected with nontarget siRNA is taken as 100%. The mean values + SEM from four independent transfection experiments with different SMCs preparations are presented. There is significant difference between the responses of SMCs transfected with anti-TPC1 siRNA and with nontarget (** p

    Journal: Cells

    Article Title: The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction

    doi: 10.3390/cells8101144

    Figure Lengend Snippet: Decrease in [Ca 2+ ] i rise in response to NE in SMCs transfected with siRNA against TPC1. ( A ) Kinetics of [Ca 2+ ] i rise in SMCs transfected with siRNA against TPC1 and nontarget siRNA. The curves from one of four transfection experiments are presented. Each point at the curves is an average of six parallel measurements. ( B ) Calcium responses to NE (100 μM) and angiotensin II (0.1 μM) in SMCs transfected with nontarget siRNA, and siRNA against TPC1 and against TPC2. [Ca 2+ ] i rise in SMCs transfected with nontarget siRNA is taken as 100%. The mean values + SEM from four independent transfection experiments with different SMCs preparations are presented. There is significant difference between the responses of SMCs transfected with anti-TPC1 siRNA and with nontarget (** p

    Article Snippet: Reagents cis -NED 19, trans-NED 19, U73122, and U73343 were acquired from Tocris (Bristol, UK); Fura2/AM, CalciumGreen/AM, and LysoTracker Red DND-99 from ThermoFischer Scientific (Waltham, MA, USA); Mitotracker Deep Red (M22426, Invitrogen), ER-tracker Red (E34250, Invitrogen), WGA Alexa Fluor 594 (Thermo Fisher, W11262), and antibodies anti-TPC1 (#ACC-071) and anti-TPC2 (#ACC-072) from Alomone Labs (Jerusalem, Israel); HRP-conjugated goat anti-rabbit IgG antibodies from BioRad (#170-6515); and the agonists of the receptors from SigmaAldrich (St. Louis, MO, USA).

    Techniques: Transfection

    Levels of TPC1 and TPC2 mRNA relative to β-actin mRNA ( A ). Western-blot of TPC1 and TPC2 proteins ( B ) isolated from rat aorta SMCs and relative levels of TPC1 and TPC2 ( C ). The values panel A are the means of four measurements in different SMCs preparation + SEM (* p

    Journal: Cells

    Article Title: The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction

    doi: 10.3390/cells8101144

    Figure Lengend Snippet: Levels of TPC1 and TPC2 mRNA relative to β-actin mRNA ( A ). Western-blot of TPC1 and TPC2 proteins ( B ) isolated from rat aorta SMCs and relative levels of TPC1 and TPC2 ( C ). The values panel A are the means of four measurements in different SMCs preparation + SEM (* p

    Article Snippet: Reagents cis -NED 19, trans-NED 19, U73122, and U73343 were acquired from Tocris (Bristol, UK); Fura2/AM, CalciumGreen/AM, and LysoTracker Red DND-99 from ThermoFischer Scientific (Waltham, MA, USA); Mitotracker Deep Red (M22426, Invitrogen), ER-tracker Red (E34250, Invitrogen), WGA Alexa Fluor 594 (Thermo Fisher, W11262), and antibodies anti-TPC1 (#ACC-071) and anti-TPC2 (#ACC-072) from Alomone Labs (Jerusalem, Israel); HRP-conjugated goat anti-rabbit IgG antibodies from BioRad (#170-6515); and the agonists of the receptors from SigmaAldrich (St. Louis, MO, USA).

    Techniques: Western Blot, Isolation