Human α‐syn (hα‐syn) was detected with a mouse monoclonal antibody (3D5; a gift from Prof. Shun Yu at Xuanwu Hospital of Capital Medical University, Beijing, China).23 Antibodies used in this study included TRPC3 (1:1000) and TRPC6 (1:500) (Alomone Lab, Jerusalem, Israel); tyrosine hydroxylase (TH) (1:2000), β‐actin (1:2000) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1:2000) (Sigma); mouse α‐syn (1:1000), caspase‐3 (1:1000) and calnexin (1:1000) (Cell Signalling Technology, Danvers, MA, USA); olfactory marker protein (Omp) (1:10,000; Wako Pure Chemical Industries, Osaka, Japan); and phosphorylated (p‐)Tyr (1:200, Santa Cruz Biotechnology). Regents used for primary neuronal culture included poly‐l ‐lysine (Sigma), Neurobasal‐A medium, 50 × B27 supplement, 0.5 mM l ‐glutamine (Gibco) and 100‐units/ml penicillin/streptomycin (Gibco).
Trpc6
Manufactured by Alomone
Sourced in Israel, United States
TRPC6 is a calcium-permeable cation channel that belongs to the transient receptor potential (TRP) channel family. It is expressed in various cell types and plays a role in the regulation of cellular calcium homeostasis and signaling processes.
Automatically generated - may contain errors
Lab products found in correlation
Trpc3, by Alomone (3 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Cal51 cells, by Leibniz Institute DSMZ (2 mentions)
Calnexin, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Tyrosine hydroxylase, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
α smooth muscle actin α sma, by Abcam (1 mentions)
Creatinine cr assay kit sarcosine oxidase, by Nanjing Jiancheng (1 mentions)
Aurka, by Proteintech (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Mk2206, by MedChemExpress (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
10 protocols using trpc6
1
Immunodetection of Human Alpha-Synuclein
2
Klotho and U73122 Modulate Fibroblast Protein Expression
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Human atrial fibroblasts with and without the administration of Klotho (100 pM, 48 h) or U73122 (1 μM, 48 h) were homogenized and centrifuged in the buffer. We separated proteins electrophoretically on a polyacrylamide gel electrophoresis with 4% to 12% sodium dodecyl sulfate and electrophoretically transferred them to a polyvinylidene difluoride membrane that was equilibrated with sodium dodecyl sulfate as described previously [21 (link)]. We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase. All bound antibodies were detected with an enhanced chemiluminescence detection system and analyzed using AlphaEaseFCTM software (Genetic Technologies, Miami, FL, USA). All targeted bands were normalized to the glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH) (Sigma-Aldrich, Merck, Darmstadt, Germany) to confirm equal protein loading.
3
Kidney Injury Mechanism Exploration
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Sources of antibodies and reagents are as follows: fetal bovine serum (FBS) (Invitrogen), DMEM/F12 (Invitrogen), anti-cleaved caspase 3 (Cat#: 9661, Cell Signaling Technology), anti-Bcl-2 (Cat#: 12789-1-AP, Proteintech), anti-Bcl-2–associated X (BAX) (Cat#: 50599-2, Proteintech), p-AKT (ser473) (Cat#: 4060P, Cell Signaling Technology), AKT (Cat#: 9272, Cell Signaling Technology), p-ERK1/2 (Cat#: 4370, Cell Signaling Technology), ERK1/2 (Cat#: 4695, Cell Signaling Technology), TRPC6 (Cat#: ACC-017, Alomone), polycystin 2 (Cat#: 19126-1-AP, Proteintech), AURKA (Cat#: 66757-1-lg, Proteintech), HIF-1α (Cat#: 79233, Cell Signaling Technology), p62 (Cat#: 88588, Cell Signaling Technology), β-actin (Cat#: TA-09, ZhongShan Biotechnology), HRP-conjugated anti-rabbit IgG (Cat#: 110777, KeRui Biotechnology), HRP-conjugated anti-mouse IgG (Cat#: 117228, KeRui Biotechnology), U0126 (Cat#: HY-12-31, MedChemExpress), MK2206 (Cat#: HY-10358, MedChemExpress), thapsigargin (Tg) (Cat#: T7459, Invitrogen), JC-1 dye (Bio-Swamp), Creatinine (Cr) Assay Kit (sarcosine oxidase) (Cat#: C011-2, Nanjing Jiancheng), Urea Assay Kit (urease) (Cat#: C013-2, Nanjing Jiancheng), and OCT compound (4583, Sakura). DMEM/F12 and FBS were purchased from Invitrogen (Chicago, California, United States). The whole sagittal section of the kidney was scanned by Biossci Biotechnology Company (Wuhan, Hubei, China).
4
Antibody and Reagent Characterization Protocol
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The following antibodies were used: TRPC6 from Alomone (ACC-017)12 (link), Sigma (PRS3889)19 (link) and SAB (21403), TRPC5 from Sigma (ACC-020)48 (link), Cav1.2 from Millipore (AB5156), Cav3.1 (ACC-021) and Cav3.3 (ACC-009) from Alomone, APP from Invitrogen (13-0200, clone LN27) and Sigma (A8717), 6E10 from Chemicon (MAB1560) and Signet (SIG-39300), MOAB-2 from Kerafast (EB2001), ADAM10 from Abcam (Ab1997), clusterin from Santa Cruz (sc-6420), BACE1 from Millipore (MAB5308), insulin-degrading enzyme from EMD Biosciences (PC730), neprilysin from Epitomics (2569–1), PS1 from Chemicon (MAB5232), nicastrin from Sigma (MAB5232), presenilin enhancer 2 from Invitrogen (36–7100), E-Cadherin from Abcam (ab53033), N-Cadherin from BD (610921), cleaved-Notch from Cell Signalling Technology (2421 L), Notch from Proteintech (10062-2-AP), EEA1 from BD (610457), Calnexin from Enzo (ADI-SPA-860-F), GM130 from BD (610823), Bip from BD (610979), Cathepsin D from Santa Cruz (sc-6487), Myc from Chemicon (05–724), HA from Sigma (H6908) and Genscript (A00168), and His from Abmart (M30111). OAG (O6754), SKF96365 (S7809) and L685,458 (L1790) were from Sigma.
5
Cardiac Protein Expression Analysis
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All protein samples were extracted from mice hearts. Protein concentrations were determined using a Bradford assay with bovine serum albumin (BSA) as a standard. To determine the protein expression of STIM1 (BD Biosciences, CA, USA), STIM2 (Cell Signaling Technology, MA, USA), TRPC1, TRPC3, TRPC4, and TRPC6 (Alomone Labs, Jerusalem, Israel), pNFATc4 (rabbit anti-mouse polyclonal antibody raised against short amino acid sequence containing Ser 168 and 170 dually phosphorylated NFATc4 of human origin, Santa Cruz Biotechnology, TX, USA), NFATc4 (rabbit anti-mouse polyclonal antibody raised against amino acids 125–198 of NFATc4 human origin, Santa Cruz Biotechnology, TX, USA), samples (50 μg) were run on 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and rocked at 4°C for 1 h in blocking buffer (0.1% Tween 20 and 1% BSA in Tris-buffered saline). An enhanced chemiluminescence (ECL)-detection system was used to detect the bound antibodies. An anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Santa Cruz Biotechnology, TX, USA) was used as an internal loading control.
6
Immunohistochemical Analysis of Dorsal Root Ganglia
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Perfusion was performed through the ascending aorta with 4% paraformaldehyde after application of sodium pentobarbital (50-mg/kg dose, i.p.). The removed DRGs were placed into 4% paraformaldehyde for post fixing overnight. Cryostat sections of 16-μm thickness were obtained, and immunohistochemistry was performed with primary antibodies for TRPC6 (1:100; Alomone Labs), PAX6 (1:200; Abcam), NF200 (1:200; Chemicon), IB4 (1:50; Sigma Aldrich), and GFAP (1:400; CST). After overnight incubation at 4°C, the sections were incubated with secondary antibodies, which conjugated with cy3 or Alex488-fluorescein isothiocyanate for 1 hour at room temperature. A Nikon (Nikon Eclipse Ni-E, Japan) confocal microscopy was used to examine the fluorescence of tissue slices.
7
Protein Extraction and Analysis of Rat DRGs
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The bilateral L4–L6 DRGs of rats were removed under sodium pentobarbital anesthesia (50 mg/kg dose, i.p.). Cytoplasm and nucleus protein of the DRGs were separated according to the manipulation of the NE-PER nuclear and cytoplasmic extraction reagents kit (ThermoFisher Scientific, Waltham, MA) (Nie et al., 2018 (link)). Briefly, the samples were homogenized on ice in a 15-mmol/L Tris buffer containing a cocktail of proteinase inhibitors and phosphatase inhibitors, and the concentration of protein was determined with a BCA protein assay (ThermoFisher Scientific). The protein samples were separated via gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. The membranes were incubated in a primary antibody against TRPC6 (1:200, Alomone Labs, Jerusalem, Israel), PAX6 (1:900, Abcam, Cambridge, UK), DNMT3b (1:500, Abcam), β-actin (1:1000, CST, Boston, MA), and histone H3 (1:1000, Abcam) overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies were used to incubate the blots. Enhanced chemiluminescence solution (Pierce) was used to detect immunocomplexes. Each band was quantified using a computer-assisted imaging analysis system (NIH ImageJ).
8
Immunofluorescence and Immunoblotting Assays
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HMLER cells were provided by R. Weinberg (Massachusetts Institute of Technology). MDA-MB-231 and HCC1806 cells were purchased from the American Type Culture Collection, and the TE3 variant of MDA-MB-231 cells was provided by S. Tavazoie (Rockefeller University). CAL-51 cells were obtained from the Leibniz Institute DSMZ, Germany. The TRPC6 inhibitor BI-749327 was purchased from MedChem Express (Catalog no. HY-111925). The paclitaxel was purchased from Selleckchem (Catalog no. S1150). The NFAT inhibitor was purchased from Cayman Chemicals (Catalog no. 13855). The following Abs that were used for flow cytometry were purchased from BioLegend: CD24–allophycocyanin (APC; ML5, BioLegend), CD44-FITC (IM7, BioLegend) and anti-rabbit FITC (MRM-47, BioLegend), anti-Rabbit-555 (Invitrogen-A32732), TRPC6 (18236–1-AP, Proteintech). For immunoblotting, the following Abs were used: Tubulin [Cell Signaling Technology (3873)], Beta-Actin (3700S, Cell Signaling Technology)TRPC6 (ACC-017, Alomone Labs), GAPDH (14C10) [Cell Signaling Technology 2118S], α6A (1A10) (MAB1356, Millipore), and α6B (6B4) (MAB1358, Millipore), Myc (Y69) (32072, Abcam), ESRP1 (PA5–25833, Thermofisher), HA (3724, Cell Signaling Technology), and GST-HRP (5475, Cell Signaling Technology). For immunofluorescence the following antibodies were used: TRPC6 (18236–1-AP, Proteintech), TAZ (clone M2–616, 560235, BD-Pharmingen).
9
Immunohistochemical Analysis of TRP Channels
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Slides were deparaffinized, rehydrated, and heated in citrate buffer pH 6 for antigenic retrieval. After blocking for endogenous peroxidase with 3% hydrogen peroxide, the following antibodies were incubated: TRPA1 (#ACC-037 1:500, 1 h, Alomone Labs, Jerusalem, Israel), TRPV2 (SAB1101376 Sigma, 1:1000, 1 h), TRPC3 (#ACC-016 Alomone, 1:500, 1 h), TRPC6 (#ACC-017 Alomone, 1:300, 1 h). Immunohistochemistry was performed using the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen (Kit LSAB, Dakocytomotion, Glostrup, Denmark). Slides were finally counterstained with haematoxylin. Negative controls were obtained after omission of the primary antibody or incubation with an irrelevant antibody. For cryo-sections, retinas were fixed overnight with 4% PFA, rinsed in PBS and then incubated with 30% sucrose before OCT embedding. Retina sections were blocked and permeabilized in PBS 0.25% triton, 5% FBS, 1% BSA for 2 h and incubated overnight at 4 °C with the rabbit anti-TRPA1 antibody (#ACC-037 Alomone, 1:100) and the rat anti-CD31 (Becton Dickinson, 1/100). After washes, sections were incubated with secondary antibody donkey anti-rabbit A488 and donkey anti-Rat A594 for 2 h at room temperature. Sections were then washed and stained with DAPI before mounting in Mowiol. Sections were imaged with a Carl Zeiss AxioImager Z1-Apotome.
10
Immunoblotting of TRPC Channels
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Rat distal de-endothelialized PA samples were sonicated in a Laemmli sample buffer that included 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 5% protease inhibitor cocktail, 2% sodium dodecyl sulfate, 1 mM EDTA and 200 μM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. As previously described, protein expression was measured by immunoblotting. 13, 15, 16 Protein samples were resolved by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and separated proteins were then transferred onto polyvinylidene difluoride membranes (pore size 0.45 μM, Bio-Rad). Membranes were blocked by 5% nonfat dry milk in Tris-buffered saline containing 0.2% Tween 20, blotted with affinity-purified rabbit polyclonal antibodies specific for TRPC1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TRPC6 (Alomone Laboratories, Jerusalem, Israel) or mouse monoclonal antibodies to α-actin (Sigma, St Louis, MO, USA) and then incubated with the appropriate secondary antibody. Blots were detected using an enhanced ECL HRP Chemiluminescent substrate reagent (Bio-Rad).
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