Journal: Circulation Research

Article Title: Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

doi: 10.1161/circresaha.115.303831

Figure Lengend Snippet: Figure 5. Transient receptor potential canonical (TRPC) channels colocalize with l-type Ca2+ channel (LTCC) in caveolae membrane microdomains where their organization is required for hypertrophic signaling. A, Plasma membranes (PMs) were purified from total cell homogenates (H) of isolated myocytes from dominant-negative TRPC4 mice. Immunoprecipitations (IPs) and Westerns were performed with the indicated antibodies (B indicates bound fraction; and U, unbound fraction). B, Sucrose density gradient fractionation on purified PMs from isolated adult feline myocytes (AFMs) infected with Ad-TRPC3 confirms the presence of LTCC and TRPC3 channels in caveolin-3 (Cav3)–enriched lipid raft membrane fractions along with the hypertrophic effector calcineurin (Cn; fraction 1 to fraction 11, F1–F11). C, AFMs were infected with Ad-NFAT-GFP and the indicated adenoviruses and NFAT translocation was monitored in response to the TRPC agonist OAG (10 umol/L) in the presence or absence of methyl-β-cyclodextrin (MβCD; 10 mmol/L). Scale bar is 10 μm. Average data are represented in D as the nuclear to cytoplasmic GFP ratio of n=100 cells per condition. P<0.05 was considered significant with *P≤0.05; **P≤0.001; ***P≤0.001 vs red fluorescent protein control; ##P≤0.001; ###P≤0.001 vs TRPC3; &&P≤0.001; &&&P≤0.001 vs TRPC6.

Article Snippet: Sheep anti-rabbit IgG-coated magnetic beads (Dynal Biotech) were pre-incubated with an antibody for TRPC4 (Alomone Labs), TRPC6 (Alomone Labs) or Cav1.2 (LTCC α1C, Millipore) for 2-4 hours at room temperature.

Techniques: Membrane, Clinical Proteomics, Purification, Isolation, Dominant Negative Mutation, Fractionation, Infection, Translocation Assay, Control

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 1. Co-immunoprecipitation of TRPV4, TRPC1 and KCa1.1(α) in the lysates of human LIMA VSMCs. A and B, TRPC1 with KCa1.1(α). C and D, TRPC1 with TRPV4. E and F, TRPV4 with KCa1.1(α). The data are representative of 3 to 4 experiments. The pulling antibody and the blotting antibody were indicated (+). Control immunoprecipitation (−) was performed using the preimmune IgG. IP, immunoprecipitation; Blot, immunoblot.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Immunoprecipitation, Control, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 5. Membrane potential changes in HEK293 cells that over-expressed with TRPV4, TRPC1 and KCa1.1. A and B, representative traces illustrating the hyperpolarizing responses to 300 nmol/L 11,12-EET (A), 5 μmol/L 4a-PDD (B) and 100 nmol/L GSK1016790A (C). D–F, summary of data showing the maximal hyperpolarization to 300 nmol/L 11,12-EET (D), 5 μmol/L 4a- PDD (E) and 100 nmol/L GSK1016790A (F) under different conditions. C1mut-pore, TRPC1 were replaced by a TRPC1 pore-dead mutant C1mut-pore. Values are means ± SE (n = 5 to 9). **, P b 0.01 compared to the controls (non-transfected); ##, P b 0.01 compared to the transfected cells without inhibitors; $$, P b 0.01 compared to preimmune IgG.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Membrane, Mutagenesis, Transfection

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 4. Requirement of TRPC1 for TRPV4 and KCa1.1 interaction. A–D, co-immunoprecipitation of TRPV4 and KCa1.1(α) in the lysates of HEK293 cells. A and B, HEK293 cells that over- expressed with TRPV4, TRPC1 and KCa1.1. C and D, HEK293 cells that over-expressed with TRPV4 and KCa1.1 but without TRPC1. The right lanes in C and D were immunoblots using cell lysates without immunoprecipitation. The pulling antibody and the blotting antibody were indicated (+). Control immunoprecipitation (−) was performed using the preimmune IgG. IP, immunoprecipitation; Blot, immunoblot. n = 3 to 4 experiments. E–F, the maximal hyperpolarization in response to 300 nmol/L 11,12-EET (E) and 5 μmol/L 4α-PDD (F) in HEK293 cells that were transfected with indicated constructs. C1 stands for TRPC1. V4 stands for TRPV4. Values are means ± SE (n = 5 to 9). **P b 0.01 compared to the controls (non-transfected), ##P b 0.01 compared to C1 + V4 + KCa1.1.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Immunoprecipitation, Western Blot, Control, Transfection, Construct

Journal: Scientific reports

Article Title: Biophysical properties of Na V 1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells.

doi: 10.1038/s41598-023-47310-6

Figure Lengend Snippet: Figure 1. Characterization of specific-markers of vCMs and aCMs. (A) qPCR analysis of several cardiomyocyte genes implicated in cellular excitability (SCN5A, CACNA1C, CACNA1D, GJA1) and contraction (TNNT2, MYL2, MYL7). The analysis of exon 25 of SCN5A mRNA covered all isoforms, including the adult and neonatal isoforms. (B) Fluorescence images showing immunolabeling of cardiac TNNT2, MYL7, MYL2, ACTN1 (α-actinin), GJA1 (connexin 43), and nuclei (DAPI, cyan) (scale bar: 40 µm). Immunofluorescence images were acquired using Zeiss LSM780 confocal microscope, processed with ZEN software (Zeiss), and adapted with ImageJ software version 1.54f (NIH, Bethesda, MD, USA). (C) Western blot analysis of the expression of several excitation–contraction coupling proteins and ion channels in vCMs and aCMs. All images of cropped blot section were exposed with an optimal time to observe protein bands. All cropped blot sections were delimited by black lines. Cropped strain-free blots showing total proteins served as loading control. Original blots are presented in Suppl. Fig. S4. Top panel, middle panel, and bottom panel, respectively, refer to “Blot 1”, Blot 2” and “Blot 3″ in Suppl. Fig. S4. Cropped section areas are indicated in Suppl. Fig. S4 by red lines. Immunoblot images were adapted with ImageJ software and arranged with Microsoft Powerpoint software version microsoft 365 (Microsoft, Redmond, WA, USA).

Article Snippet: The PVDF membranes were blocked and were incubated with rabbit anti-sodium voltage-gated channel alpha subunit 5 (SCN5A) (1:200, Cat# ASC-005, Alomone Labs, RRID:AB_2040001), rabbit anti-calcium voltagegated channel subunit alpha 1 C (CACNA1C) (1:200, Cat# ACC-003, Alomone Labs, RRID:AB_2039771), mouse anti-myosin light chain 7 (MYL7) (1:400, Cat# ab68086, Abcam, RRID:AB_1140497), rabbit anti-myosin light chain 2 (MYL2) (1:2000, Cat# ab79935, Abcam, RRID:AB_1952220), mouse anti-TNNT2 (1:5000, Cat# ab10214, Abcam, RRID:AB_2206574), rabbit anti-gap junction protein alpha 1 (GJA1) (1:5000, Cat# ab11370, Abcam, RRID: AB_297976), rabbit anti-potassium voltage-gated channel subfamily A member 5 (KCNA5) (1:200, Cat# APC-150, Alomone Labs, RRID: AB_10918640), rabbit anti-ryanodine receptor 2 (RYR2) (1:1000, Cat# ARR002, Alomone Labs, RRID: AB_2040184), or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:20,000, Cat# A300-641A, Bethyl, RRID:AB_513619).

Techniques: Fluorescence, Immunolabeling, Immunofluorescence, Microscopy, Software, Western Blot, Expressing, Control