Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a Venn diagram showing the overlap of genes upregulated in high versus low chemotherapy-resistant cervical cancer patients (red), CaSki NANOG versus CaSki no insert (blue), and cervical cancer patients from the TCGA cohort classified as having high or low EGFR activity score (dark). b The cells were stained with anti-TRPV1 (red) antibodies and then visualized by confocal microscopy. DAPI was used to stain the nuclei. Scale bar, 10 μm. The graph depicts the experimental quantitation of TRPV1. The protein levels of TRPV1 and β-actin were confirmed by western blots. c Current-voltage relationships from capsaicin (Cap, red line) or AMG9810 (AMG, blue line)-induced whole-cell current in CaSki P versus CR cells. Summary of average outward current at +60 mV during Cap exposure in CaSki P versus CR cells. d The protein levels of secreted EGF and internal LC3B, pEGFR, and EGFR were confirmed by western blots. e Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. f mRNA expression of TRPV1 was analyzed by qRT-PCR. g TRPV1 mRNA levels were measured by qRT-PCR. h Levels of TRPV1 and FLAG-NANOG proteins were confirmed by western blot. i Diagram of the human TRPV1 promoter region containing the NANOG-binding site. The arrows indicate the qChIP-PCR amplicon. j The cross-linked chromatin from HEK293 cells transfected with empty vector or FLAG- NANOG was immunoprecipitated with rabbit IgG or anti-FLAG antibodies. Relative enrichment of FLAG-NANOG in the TRPV1 promoter region was assessed by qChIP-PCR analysis with primers that amplified the genomic region indicated above. The ChIP data values represent the relative ratio to the input. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-tailed Student’s t test ( b ), two-way ANOVA ( c , e ), or one-way ANOVA ( f , g , j ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file. (NS, not significant).

Article Snippet: The sections were incubated with rabbit polyclonal anti-TRPV1 antibodies (Alomone, Jerusalem, ISR, ACC-030) at 1:1000 dilution for 60 min.

Techniques: Activity Assay, Staining, Confocal Microscopy, Quantitation Assay, Western Blot, Flow Cytometry, Incubation, Expressing, Quantitative RT-PCR, Binding Assay, Amplification, Transfection, Plasmid Preparation, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a , b CaSki NANOG cells were transfected with siRNA targeting GFP or TRPV1 . c , d CaSki NANOG cells were treated with DMSO or AMG9810. a , c The levels of TRPV1, LC3B, pEGFR, EGFR, pAKT, and AKT proteins were confirmed by western blot analysis. b , d The frequency of apoptotic (active caspase 3 + ) cells were analyzed by flow cytometry. e Current-voltage relationships in capsaicin (Cap, red line) or AMG9810 (AMG, blue line)-induced whole-cell current in CaSki no insert versus CaSki NANOG cells. f Fluorescence calcium imaging by TRPV1 activity in tumor cells. Top, representative calcium images of FURA-2-AM-loaded cells. Bottom, changes in intracellular Ca 2+ current over time in tumor cells. g , h CaSki NANOG cells were transfected with siRNA targeting GFP or CaMKKβ . The expression levels of indicated protein were confirmed by western blots. a , c , g , h β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-way ANOVA ( b , d ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: The sections were incubated with rabbit polyclonal anti-TRPV1 antibodies (Alomone, Jerusalem, ISR, ACC-030) at 1:1000 dilution for 60 min.

Techniques: Transfection, Western Blot, Flow Cytometry, Fluorescence, Imaging, Activity Assay, Expressing

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a Representative image of immunohistochemical staining in chemoradiosensitive and chemoradioresistant patient specimens. The boxed regions are displayed at high magnification in the inset. Scale bar, 100 µm. b Box plot depiction of NANOG, TRPV1, and pEGFR protein expression in chemoradiosensitive ( n = 43) and chemoradioresistant ( n = 21) cervical cancer patients. c Combinations of NANOG, TRPV1, and pEGFR expression were compared between chemoradiosensitive and chemoradioresistant cervical cancer cases. d Kaplan–Meier plots of overall survival according to combinations of NANOG, TRPV1, and pEGFR expression. In the box plots, the top and bottom edges of boxes indicate the first and third quartiles, respectively; the center lines indicate the medians; and the ends of whiskers indicate the maximum and minimum values, respectively. The p values by two-tailed Student’s t test ( b ), Mann–Whitney U test ( c ), or Gehan–Breslow–Wilcoxon test ( d ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file. (NS, not significant).

Article Snippet: The sections were incubated with rabbit polyclonal anti-TRPV1 antibodies (Alomone, Jerusalem, ISR, ACC-030) at 1:1000 dilution for 60 min.

Techniques: Immunohistochemical staining, Staining, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a – c CaSki no insert and TRPV1 cells were treated with DMSO or AMG9810, as indicated. a The protein levels of TRPV1, LC3B, pEGFR, and EGFR were confirmed by western blots. b The frequency of apoptotic (active caspase 3 + ) cells were analyzed by flow cytometry. c The amount of EGF secreted into the media was measured by ELISA. d CaSki TRPV1 cells were treated with IgG or anti-EGF. Levels of pEGFR and EGFR were confirmed by western blots. e and f CaSki TRPV1 cells were transfected with siRNA targeting GFP or EGFR . e The protein levels of pEGFR and EGFR were confirmed by western blot analysis. f Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. a , d , e β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-way ANOVA ( b , f ) or one-way ANOVA ( c ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file. (NS, not significant).

Article Snippet: The sections were incubated with rabbit polyclonal anti-TRPV1 antibodies (Alomone, Jerusalem, ISR, ACC-030) at 1:1000 dilution for 60 min.

Techniques: Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transfection, Incubation, Expressing

Journal: PLoS ONE

Article Title: N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

doi: 10.1371/journal.pone.0043525

Figure Lengend Snippet: A. Cryostat sections of rat renal tissue were stained for TRPV1 by indirect immunofluorescence using polyclonal an antibody (left). Note that distinct tubular structures were positive for TRPV1 (arrows). Omission of the primary was used as negative control (right). B. At higher magnifications it was observed that PGP9.5 positive nerve fibers (arrows) within the rat kidney also express the capsaicin receptor TRPV1 as shown by the overlay of both.

Article Snippet: TRPV1 in frozen kidney sections was detected using rabbit anti-TRPV1 polyclonal primary antibody (Alomone) and secondary anti-rabbit Cy3 (Jackson ImmunoResearch, PA, USA).

Techniques: Staining, Immunofluorescence, Negative Control

Journal: PLoS ONE

Article Title: N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

doi: 10.1371/journal.pone.0043525

Figure Lengend Snippet: A. Increases in free intracellular calcium in response to repetitive application of NOD displayed marked tachyphylaxis from stimulus to stimulus (grey trace). Application of the competitive TRPV1 antagonist capsazepine before and during the 2 nd stimulus (CPZ; black trace) abolished the NOD response. B. Mean change in intracellular calcium by NOD in cells challenged with (filled bars; n = 4) and without CPZ during the second stimulus (open bars; n = 5) indicated the specificity of NOD at TRPV1. The threshold for a significant response is indicated by the dotted line (*p<0.05, ***p<0.001 vehicle versus CPZ, student’s unpaired t-test).

Article Snippet: TRPV1 in frozen kidney sections was detected using rabbit anti-TRPV1 polyclonal primary antibody (Alomone) and secondary anti-rabbit Cy3 (Jackson ImmunoResearch, PA, USA).

Techniques:

Journal: PLoS ONE

Article Title: N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

doi: 10.1371/journal.pone.0043525

Figure Lengend Snippet: A. Human PTECs were marked with DAPI and stained with a primary anti-TRPV1 antibody (giunea-pig polyclonal serum); omission of a primary antibody served as negative control ( B ). C. In addition TRPV1 was assessed in lysates of unstimulated (1) or TNF-α stimulated PTECs (2) by Western blotting. TRPV1 transfected HEK cells (3) were used as positive control to indicate the molecular weight of TRPV1.

Article Snippet: TRPV1 in frozen kidney sections was detected using rabbit anti-TRPV1 polyclonal primary antibody (Alomone) and secondary anti-rabbit Cy3 (Jackson ImmunoResearch, PA, USA).

Techniques: Staining, Negative Control, Western Blot, Transfection, Positive Control, Molecular Weight

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: (A) Immunolabeling of the neuronal marker UCHL1 and TRPV1 in frozen DRG sections and cultured sensory neurons from rat. TRPV1 is selectively expressed in a subpopulation of sensory neurons. (B) Representative view field showing the automated image analysis to quantify TRPV1 expression in cultured sensory neurons. Green encircled objects represent sensory neurons marked by UCHL1. (C) Distribution of TRPV1 immunofluorescence intensities in frozen DRG sections (red line) and cultured sensory neurons (blue line). The scattered line indicates the threshold used to determine the number of TRPV1(+) neurons. (D) Work flow to remove TRPV1(+) neurons from freshly isolated sensory neurons. (E) Immunoblot showing the reduction of TRPV1 protein following depletion of TRPV1(+) neurons with 10 µM capsaicin for 30–120 min. (F) Time-dependent reduction of TRPV1 mRNA after removal of TRPV1(+) neurons with 10 µM capsaicin or 100 nM RTX determined by qPCR. (G) Dose-dependent reduction of TRPV1 mRNA following depletion of TRPV1(+) neurons with RTX for 30 min determined by qPCR. (H) Agonist-treated neurons were cultured overnight, immunostained for TRPV1, and analyzed by quantitative microscopy. Both agonists effectively reduced the number of TRPV1(+) neurons. Scattered lines indicate the threshold used to determine the number of TRPV1(+) neurons. (I) Quantification of TRPV1(+) neurons following treatment with 10 µM Cap or 100 nm RTX, respectively (n = 3, p<0.01, one-way ANOVA with Bonferroni's multiple comparisons test).

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Immunolabeling, Marker, Cell Culture, Expressing, Immunofluorescence, Isolation, Western Blot, Microscopy

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: (A) qPCR quantification of TRPV1 mRNA levels in samples hybridized on microarrays (n = 4, p<0.001, One-way ANOVA). (B) Relative expression levels of TRPV1 mRNA determined by microarray hybridizations (n = 4, p<0.001, One-way ANOVA with Bonferroni's multiple comparisons test). (C) Correlation matrix of the expression level of all genes (normalized fluorescence intensities) detected using micorarrays. Clustering of overall gene expression is visible between RTX and capsaicin treated samples. (D) qPCR quantification of TRPV1 mRNA levels in samples used for RNA-Seq (n = 3, p<0.001, paired two-tailed t-test). (E) Relative expression levels of TRPV1 mRNA determined by RNA-Seq (n = 3, p<0.001, One-way ANOVA). (F) Overview of the number of target transcripts identified by microarray hybridizations and RNA-Seq. Raw fluorescence intensities of microarrays were background corrected, log2 transformed, normalized, and filtered for expressed genes (Illumina detection p-value <0.01 in at least one of the samples, see sections for details). Sequencing was performed using a 5500xl SOLiD System resulting in 664 million reads in total (see sections for details). The reads were filtered to remove ribosomal RNA, tRNAs, and vector sequences. The remaining reads mapped to 16590 genes of the reference genome (rn5). Read counts were transformed to RPKM values (Reads per kilo base per million), normalized, and filtered to remove weakly expressed transcripts (RPKM>0.1). P-values of differentially expressed genes identified with both methods were adjusted for multiple testing with Benjamini and Hochberg's method, adjusted p-values <0.05 were considered significant.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Microarray, Fluorescence, RNA Sequencing Assay, Two Tailed Test, Transformation Assay, Sequencing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: (A) Correlation of fold-changes obtained by microarray hybridizations and RNA-Seq (Spearmans ρ = 0.66, p<0.0001). (B) qPCR validation of 14 transcripts identified as differentially expressed by microarray hybridizations or RNA-Seq. The depletion of TRPV1(+) neurons was performed with capsaicin (1 and 10 µM) for 30 min. The reduction of all target transcripts is dose-dependent.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Microarray, RNA Sequencing Assay

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: Top 50 transcripts found with higher expression levels in TRPV1(+) neurons by microarrays and/or RNA-Seq (adj. p <0.05, fold-change> 1.5, RPKM> 0.5).

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: (A) Triple staining of the neuronal marker UCHL1 and two different TRPV1 antibodies derived from rabbit and goat, respectively, to facilitate the analysis of various targets. The staining intensities obtained with both TRPV1 antibodies correlated significantly (Spearmans ρ = 0.96, p<2.2e-16). (B-E, G) Co-labeling of TRPV1 and CART (B), Nos1 (C), KChIP1 (D), KChIP2 (E), and CaMKIIα (G). Plots of respective controls are shown in . (F) Average fluorescence intensities of TRPV1 and the indicated targets in TRPV1-negative (grey) and -positive (black) neurons. Signal intensities of all analyzed targets were significantly higher within the TRPV1(+) population (n = 3 with>3000 analyzed neurons per experiment, paired two-tailed t-tests).

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Staining, Marker, Derivative Assay, Labeling, Fluorescence, Two Tailed Test

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: (A) Dose-dependent induction of RII phosphorylation in sensory neurons after 1 min stimulation with PGD 2 (EC 50 = 377 nM, n = 3,>2000 neurons/condition; one-way ANOVA with Bonferroni's multiple comparisons test). (B) PGD 2 did not induce pRII in non-neuronal cells of the same cultures shown in A. (C) Time course of RII phosphorylation indicating long-lasting effects of PGD 2 (10 µM) on sensory neurons. (D) Stimulation with PGD 2 also results in phosphorylation of the ERK1/2 measured in the same cultures shown in D. (E) Representative experiment demonstrating that induction of RII phosphorylation is enhanced in TRPV1(+) neurons (total of 3664 neurons). Plots of respective controls are shown in . (F) Fold changes of pRII intensities in TRPV1(−) (grey bars) and TRPV1(+) (black bars) neurons after 1 min stimulation with 10 µM PGD 2 (n = 3,>2000 neurons/condition, one-way ANOVA with Bonferroni's multiple comparisons test). (G) Co-labeling of TRPV1 and PTGDS revealing that PTGDS is expressed in neurons lacking TRPV1 (total of 9951 neurons, also refer to . for control plots). (H) Co-labeling of NF200 and PTGDS showing that PTGDS(+) neurons express NF200 (total of 12966 neurons, also refer to . for control plots).(I) Size distribution of PTGDS(+) (green), NF200(+) (red), and all sensory neurons (black) indicating that PTGDS(+) neurons are larger than other neurons. (J) Suggested pathway of interneuronal communication between subgroups of sensory neurons. Large-diameter mechanosensitive neurons express PTGDS resulting in the production of PGD 2 , which activates DP1 receptors present on nociceptive neurons expressing TRPV1.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Labeling, Expressing

Journal: PLoS ONE

Article Title: The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

doi: 10.1371/journal.pone.0057149

Figure Lengend Snippet: A. Semi-quantitative RT-PCR for TRPV1. Upper left panel: mRNA levels from PC3 prostate cancer cells: non treated cells, cells treated with 32 µM capsaicin for 1 hr and cells treated with heat shock at 42°C for 3 hrs. Lower right panel: mRNA levels of GAPDH as well as densitometric. Upper right panel: mRNA levels from HEK-293e cells: non treated cells, cells treated with 32 µM capsaicin for 1 hr (Caps), cells treated with heat shock at 42°C for 3 hrs, and cells treated with heat shock at 42°C for 3 hrs with the addition of 32 µM capsaicin. Lower right Panel: mRNA levels of GAPDH as well as densitometry. B. Representative immunoblot analysis of TRPV1 abundance. TRPV1 was detected in HEK-293e cells. Cells in culture were incubated with 0.01% DMSO or treated with 4 µM, 16 µM or 32 µM of capsaicin for 1 hr. C. Representative immunoblot analysis of TRPV1 and Hsp90 abundances in HEK-293e cells. Cells were treated with 32 µM capsaicin for 0 hr, 1 hr, 4 hrs and 6 hrs.

Article Snippet: TRPV1 rabbit polyclonal antibody 1∶200 (Alomone Labs Inc., Israel) and Hsp70 mouse monoclonal 1∶50 (Stressgen, Biotechnologies Corp., Canada) were added for an hour.

Techniques: Quantitative RT-PCR, Western Blot, Incubation

Journal: PLoS ONE

Article Title: The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

doi: 10.1371/journal.pone.0057149

Figure Lengend Snippet: A. Left panel: Double immunofluorescence staining of TRPV1 and Hsp70 in HEK-293e cells treated with 32 µM capsaicin for 1 hr. TRPV1 visualized in green, Hsp70 in red and DAPI (Nuclei) in blue. White arrows indicate Hsp70 abundance (left picture) and co-localization (right picture). B. Right panel: Immunofluorescence staining of Hsp70 in HEK-293e cells in non-treated cells, treated cells with 32 µM of capsaicin for 1 hr, heat shock treated cells for 3 hrs at 42C and cells transfected with 200 nM TRPV1 siRNA and treated with heat shock for 3 hrs at 42C.

Article Snippet: TRPV1 rabbit polyclonal antibody 1∶200 (Alomone Labs Inc., Israel) and Hsp70 mouse monoclonal 1∶50 (Stressgen, Biotechnologies Corp., Canada) were added for an hour.

Techniques: Double Immunofluorescence Staining, Immunofluorescence, Staining, Transfection

Journal: PLoS ONE

Article Title: The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

doi: 10.1371/journal.pone.0057149

Figure Lengend Snippet: A. Representative immunoblot analysis of Hsp70 and TRPV1 in HEK with or without the transfection of the TRPV1 gene (HEK, HEK+V1) and S2 drosophila cells lacking the TRPV1 gene (S2). B. Semi-quantitative RT-PCR for TRPV4. Upper panel: mRNA levels from HEK and PC3 prostate cancer cells: non treated cells (DM), cells treated with 32 µM capsaicin for 1 hr (Cps) and cells treated with heat shock at 42°C for 3 hrs (HS) or with heat shock at 42°C for 3 hrs with the addition of capsaicin (Cps+HS). Lower panels: mRNA levels from HEK-293e cells: non treated cells (DM), cells treated with 32 µM capsaicin for 1 hr (Cps), cells treated with heat shock at 42°C for 3 hrs (HS), and cells treated with heat shock at 42°C for 3 hrs with the addition of 32 µM (Cps+HS) and mRNA levels of GAPDH.

Article Snippet: TRPV1 rabbit polyclonal antibody 1∶200 (Alomone Labs Inc., Israel) and Hsp70 mouse monoclonal 1∶50 (Stressgen, Biotechnologies Corp., Canada) were added for an hour.

Techniques: Western Blot, Transfection, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

doi: 10.1371/journal.pone.0057149

Figure Lengend Snippet: A. Hsp70 co-localized in MCF-7 cell membrane. Immunofluorescence staining of Hsp70 in MCF-7 cells treated with 32 µM capsaicin for 1 hr. Hsp70 visualized in red and DAPI (Nuclei) in blue. White arrows indicate co-localization of Hsp70 to the membrane (right picture). B. Hsp70 expression in MCF-7 cells. Immunofluorescence staining of Hsp70 in MCF-7 cells in non treated cells compared to treated cells with 32 µM capsaicin for 1 hr. C. TRPV1 expression in lung homogenate in vivo . Representative immunoblot analysis of TRPV1 and Hsp70 abundances. TRPV1 and Hsp70 were detected in lungs of untreated rats (T0), septic rats treated with PBS (2CLPPBS) or septic rats treated with adenovirus vector over-expressing Hsp70 (2CLPAdHSP). The numbers (1, 2) represent repeated experiments. D. Co-IPs studies in lung homogenates treated with adenoviral vector over-expressing Hsp70 in-vivo . 100 µg of lung homogenates were Co-IPed with TRPV1. Representative immunoblot analysis of TRPV1 and Hsp70 abundances.

Article Snippet: TRPV1 rabbit polyclonal antibody 1∶200 (Alomone Labs Inc., Israel) and Hsp70 mouse monoclonal 1∶50 (Stressgen, Biotechnologies Corp., Canada) were added for an hour.

Techniques: Immunofluorescence, Staining, Expressing, In Vivo, Western Blot, Plasmid Preparation

Journal: PLoS ONE

Article Title: The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

doi: 10.1371/journal.pone.0057149

Figure Lengend Snippet: Upper panels: A. TRPV1 siRNA – sequence No. 1: Knockdown of TRPV1, using siRNA, in HEK-293 and HT-29 cells resulted in inhibition of Hsp90, and Hsp70 expression. HEK293 & HT-29 cells were treated with either oligofectamine alone, heat shock at 42°C for 3 hrs, heat shock at 42°C for 3 hrs with the transfection of 100 nM TRPV1 siRNA or with the transfection of 200 nM TRPV1 siRNA. Cells were transfected with TRPV1 siRNA for 72 hrs, prior to heat shock. Lanes grouped by curly brackets represent repeated RNAi experiments. B. TRPV1 siRNA – sequence No. 2 & scrambled siRNA: Knockdown of TRPV1, using a second sequence of siRNA and 200 nM of scrambled siRNA in HEK-293 and HT-29 cells, resulted in inhibition of Hsp90, and Hsp70 expression.Lower panel: Representative immunoblot of TRPV1 abundance. TRPV1 was detected in HEK-293 cells treated with heat shock at 42°C for 3 hrs with the transfection of either 100 nM TRPV1 siRNA or 200 nM TRPV1 siRNA. Lanes grouped by curly brackets represent repeated RNAi experiments.

Article Snippet: TRPV1 rabbit polyclonal antibody 1∶200 (Alomone Labs Inc., Israel) and Hsp70 mouse monoclonal 1∶50 (Stressgen, Biotechnologies Corp., Canada) were added for an hour.

Techniques: Sequencing, Inhibition, Expressing, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Mechanisms Underlying Ectopic Persistent Tooth-Pulp Pain following Pulpal Inflammation

doi: 10.1371/journal.pone.0052840

Figure Lengend Snippet: A: TRPV1-IR cells (upper panel), NeuN-IR cells (middle panel) and merged photomicrographs (lower panel) in M2 capsaicin-applied rats which received CFA to M1. B-G: FG(+) TG cells expressing TRPV1-IR in M1saline-adminstrated (B, C and D) and M1 CFA-administrated (E, F and G) rats. H: The mean number of FG(+) TRPV1-IR cells/FG (+) following saline (open bur) or CFA (solid bur) application to M1. I: The mean number of FG(+) TRPV1-IR cells/FG(+) following Veh (solid bur) or FC (open bur) injection into TG in M1 CFA rats. *: p<0.05.

Article Snippet: Sections were incubated with rabbit anti-TRPV1 polyclonal antibody (1∶500; Alomone labs, Jerusalem, Israel), in 0.01 M PBS containing 4% NGS and 0.3% Triton X-100 (Sigma-Aldrich) overnight at 4°C.

Techniques: Expressing, Injection

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) PHluorin was inserted into the turret region of the TRPV1 channel, between residues H614 and K615 using CRISPR/Cas9 technology ( , ). ( B ) Representative confocal images showing GFP expression in DRG, nodose ganglion (NG), and spinal cords of WT and TRPV1-pHluorin mouse. Scale bars: 50 μm (DRG and nodose ganglion); 100 μm (spinal cord). ( C ) Coimmunostaining of GFP and TRPV1 in DRG; coimmunostaining of GFP with markers of peptidergic (CGRP) or nonpeptidergic (IB4) neurons in the spinal dorsal horn. Scale bars: 50 μm (DRG); 100 μm (spinal cord). ( D ) Western blot of DRG lysates from WT and TRPV1-pHluorin mice using an anti-GFP antibody; note the specific band at approximately 125 kDa only in the transgenic mice. Immunoprecipitation of TRPV1-pHluorin from DRG lysates using GFP-trap. Membranes were then probed with an anti-TRPV1 antibody. Note the absence of band in WT animals. ( E ) Representative whole-cell current-clamp recordings of the capsaicin-evoked AP discharge in DRG neurons from WT and TRPV1-pHluorin mice. ( F ) Dose-response curve evoked by capsaicin, measured by calcium imaging on DRG neurons from WT (circles) (EC 50 = 0.91 ± 0.12 μM, n = 34) or TRPV1-pHluorin (pHluo) mice (squares) (EC 50 = 1.06 ± 0.09 μM, n = 28) at RT (22°C). Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: CRISPR, Expressing, Western Blot, Transgenic Assay, Immunoprecipitation, Imaging

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Experimental approach used to conduct the microarray analysis from TRPV1 neurons, 72 hours after i.pl. CFA. ( B ) FACS isolation of TRPV1-pHluorin neurons. Representative FACS plot of GFP + population in WT (top) and TRPV1-pHluorin (bottom) mice. SSC-A side scatter area; FSCA- forward scatter area. ( C ) Scatter plot representation of genes regulated in CFA conditions. Genes that passed a threshold of log 2 fold change in differential expression analysis are represented as green when downregulated and red when upregulated. All genes are listed in . ( D ) qRT-PCR assessment of ALKAL2 upregulation in the DRG ipsilateral to the CFA injection (Ipsi) ( n = 9), compared with the contralateral side (Contra) ( n = 9) and naive control ( n = 8). Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s post hoc test. * P < 0.05; *** P < 0.001. ( E ) Representative Western blot of ALKAL2 in the DRG ipsilateral to the CFA injection compared with the contralateral side. ( F ) Quantification of ALKAL2 protein level from Western blot experiments. Each dot represents a sample collected from a different animal ( n = 6 per group). Statistical analysis was performed by unpaired t test ( F ). **** P < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Microarray, Isolation, Expressing, Quantitative RT-PCR, Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Heatmap of the expression of ALKAL2 and selected population markers on the 17 populations of sensory neurons from DRG described in Zeisel et al. . ( B ) Representative confocal images of coimmunostaining for ALKAL2 and TRPV1, IB4, GFRα3, and NF200 in DRG neurons. Scale bars: 50 μm. ( C ) Dot plot summarizing the results in B : 77.64% ± 2.95% of TRPV1, 77.64% ± 3.06% of GFRα3, 41.65% ± 7.08% of IB4, and 36.46% ± 2.64% of NF200-positive neurons express ALKAL2 (each symbol represents a DRG section from n = 4 individual animals). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. **** P < 0.001. ( D ) Representative confocal images of coimmunostaining for ALKAL2 and NF200, IB4, and GFRα3 in the sciatic nerve. Scale bars: 50 μm. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Schematic illustrating the coculture system used to chronically expose DRG neurons to ALKAL2. HEK cells were plated into the upper chamber of a Transwell and then transfected with ALKAL2 plasmid for 16 hours. Cells were washed, and DRG neurons were plated in the lower chamber of the Transwell for another 16 hours of coculture and then immunostained for Tuj1. Scale bars: 50 μm. ALKAL2 induces a significant increase of total neurites ( B ), number of branch points per neuron ( C ), and total neurite length per neuron ( D ) (<20 μm). Control, n = 19; ALKAL2, n = 12; ALKAL2+lorlatinib, n = 18. Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s post hoc test. * P < 0.05; ** P < 0.01; **** P < 0.001. ( E ) Representative confocal images illustrating the TRPV1-GFP innervation of the skin paw following i.pl. injection of CFA (3 days). Scale bars: 50 μm. ( F ) CFA-induced sprouting is reversed by daily administration of lorlatinib (1 mg/kg). Control, n = 5; CFA+vehicle, n = 5; CFA+lorlatinib, n = 5. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; ** P < 0.01. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Transfection, Plasmid Preparation, Injection

Journal: Cell Journal (Yakhteh)

Article Title: Expression of TRPV1 as A Heat Sensitive Voltage-Dependent Ion Channel and Oxidative Stress in Sperm Samples of Infertile Men with Varicocele: A Case-Control Study

doi: 10.22074/cellj.2022.8038

Figure Lengend Snippet: Assessment of sperm TRPV1 at RNA and protein levels. A. Comparison of mean percentage (P=0.001) and intensity of TRPV1 (In-TRPV1) proteins between varicocele (n=25) and fertile groups (n=25). In-TRPV1 show relative fluorescence intensity of TRPV1 in sperm sample. B. Flow cytometric plot of TRPV1 protein in a fertile man and C. An infertile man with varicocele. D. Comparison of gene expression analysis of the TRPV1 gene relative to GAPDH gene (housekeeping gene as an internal control) between fertile and varicocele groups. E. Localization of TRPV1 protein in the sperms of fertile individuals. Values are expressed as mean ± standard error of the mean (SEM). The significant difference is presented as ** P<0.01.

Article Snippet: Following the centrifugation at 3000 rpm and removal of the supernatant, test and control tubes were incubated overnight at 4˚C with rabbit polyclonal anti-TRPV1 antibody (Cat No:ACC030, Alomone labs, Jerusalem BioPark, Israel) and 1% BSA solution, respectively.

Techniques: Fluorescence, Expressing

Journal: Cell Journal (Yakhteh)

Article Title: Expression of TRPV1 as A Heat Sensitive Voltage-Dependent Ion Channel and Oxidative Stress in Sperm Samples of Infertile Men with Varicocele: A Case-Control Study

doi: 10.22074/cellj.2022.8038

Figure Lengend Snippet: Comparison of TRPV1 protein localization between fertile and varicocele groups, before and after induction of acrosome reaction using the calcium ionophore at A. Acrosomal and B. Equatorial region. Values are expressed as mean ± standard error of the mean (SEM). The significant difference is presented as *; P<0.05 and **; P<0.01. A/R; Acrosom reaction.

Article Snippet: Following the centrifugation at 3000 rpm and removal of the supernatant, test and control tubes were incubated overnight at 4˚C with rabbit polyclonal anti-TRPV1 antibody (Cat No:ACC030, Alomone labs, Jerusalem BioPark, Israel) and 1% BSA solution, respectively.

Techniques: