rabbit polyclonal anti-trpc1 antibody  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Selective Assembly of TRPC Channels in the Rat Retina during Photoreceptor Degeneration

doi: 10.3390/ijms25137251

Figure Lengend Snippet: Features of the primary antibodies.

Article Snippet: TRPC1 , Intracellular aa’ of human TRPC1 , Rabbit Polyclonal , Alomone Labs (Jerusalem, Israel, #ACC-010 , IC: 1:500 PLA 3 : 1:400.

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: The store-operated Ca 2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

doi: 10.1016/j.jbc.2023.102882

Figure Lengend Snippet: TRPC1 and Orai3 are involved in carbachol (CCh)-evoked NF-κB activation in Orai1-KO cells. A, Orai1-KO HEK-293 cells (O1KO) transfected with either shTRPC1 (O1KO (shT1)), shTRPC6 (O1KO (shT6)), shOrai3 (O1KO (shO3)), or scramble plasmid (O1KO) were transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and then stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity of the lysates was measured as described in the section. The luminescence in relative light units (RLUs) of unstimulated O1KO, O1KO(shT1), O1KO(shT6), and O1KO(shO3) HEK-293 cells were 598,259 ± 47,521, 601,025 ± 51,248, 599,987 ± 32,581, and 601,277 ± 39,985, respectively). B, O1KO, O1KO (shT1), and O1KO (shT6) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-TRPC1 ( left panels ) or anti-TRPC6 ( right panels ) antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of TRPC1 and TRPC6 bands was normalized to β-actin and quantified ( bar graph ). C, WT HEK-293 cells (WT), WT cells transfected with shOrai3 (shO3), O1KO cells, O1KO cells transfected with shOrai3 (O1KO (shO3)), and Orai1/Orai2/Orai3 triple-KO cells (TKO) transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity was measured as described in the section. The luminescence in RLUs of unstimulated WT, O1KO, O1KO(shO3), and TKO HEK-293 cells were 610,982 ± 48,875, 607,588 ± 47,510, 592,115 ± 37,514, and 597,323 ± 37,899, respectively. D, WT HEK-293 cells (WT) and WT cells transfected with shOrai3 (shO3) were lysed and subjected to 10% SDS-PAGE and Western blotting with anti-Orai3 antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). E, WT, O1KO, TKO, and O1KO (shO3) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-Orai3. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). For A, from left to right , n = 18, 14, 18, 13, 18, 12, 18, and 13; for C, from left to right , n = 30, 24, 12, 12, 18, 13, 18, 14, 18, and 12; n values correspond to separate experiments. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ∗ p < 0.05 and ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as compared with CCh-treated O1KO HEK-293 cells ( A ) or CCh-treated WT HEK-293 cells ( C ). $ p < 0.05, $$ p < 0.01, and $$$$ p < 0 .0001 as compared with their respective control (untreated) cells. HBS, Hepes-buffered saline; HEK-293, human embryonic kidney 293 cell line.

Article Snippet: High-glucose Dulbecco’s modified Eagle’s medium, fetal bovine serum, trypsin, penicillin/streptomycin, TRIzol reagent, qRT–PCR primers, high-capacity complementary DNA reverse transcription kit, SYBR Green PowerUp, rabbit polyclonal anti-TRPC1 antibody (catalog number: PA577303, epitope: amino acids 557–571 of human TRPC1), Clean-Blot IP detection reagent, and SuperSignal West Dura extended duration substrate reagent, and Pierce BCA protein assay kit were purchased from Thermo Fisher Scientific.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, SDS Page, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: Sites where STIM1 and TRPC1 coincide with other proteins in untreated cells. Loci with colocalized proteins are indicated by yellow arrowheads. Coincidence of the labels in higher parts of the cell was ignored because of the addition of fluorescent signals originating from deeper levels in the cell. a STIM1 shows little tendency to colocalize with Orai1 or TRPC1 but consistent colocalization with AQP4. TRPC1 is found in linear arrays (black arrows). AQP4 appears in loci of two distinct size classes, with the smaller confined to the cell edge (green bracket). b TRPC1-containing loci contain Orai1 and AQP4 but infrequently the VACC channel subunit, CaV1.2. Areas where the red and green images coincide are mainly in thick portions of the cell where loci are superimposed due to overlapping layers of structure. TRPC1 coincides with Vamp2 in some sites (yellow arrowheads), but in others, diffuse Vamp2 encircles the TRPC1 sites (orange arrowheads). ( b ) TRPC1-containing loci contain Orai1 and AQP4 but infrequently the VACC channel subunit, CaV1.2. Areas where the red and green images coincide are mainly in thick portions of the cell where loci are superimposed due to overlapping layers of structure. TRPC1 coincides with Vamp2 in some sites (yellow arrowheads), but in others, diffuse Vamp2 encircles the TRPC1 sites (orange arrowheads). Bars = 10 µm

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: Correlation coefficients for proteins paired with STIM1 and TRPC1 in untreated cells

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: TRPC1 on microtubules and filopodia. a Linear arrays of TRPC1 (green) are aligned parallel to features containing STIM1 (red). Inset: enlargement of area designated by arrowhead, showing features side by side. b TRPC1 loci aligned on filopodia (white arrowheads). c TRPC1 loci (green arrowheads) alternating with STIM1 (red) during Ca 2+ readdition. d Colocalization of β-tubulin (left panel) and TRPC1 (middle panel) in an untreated cell. TRPC1 colocalizes with microtubules bordering the edge (arrowheads) and in the interior (arrows). Panel at right is the overlay. e Microtubules (red) labelled by antibody against β-tubulin penetrate to the cell edge but do not enter filopodia (white arrowheads) in an untreated cell. The colocalization of TRPC1 and β-tubulin is apparent in higher parts of the cell (black arrow). Inset: TRPC1 loci (green) free of microtubules (red) at the cell edge. f Colocalization of β-tubulin (upper frame) and TRPC1 (lower frame) shows that TRPC1 spots (green arrowheads) extend beyond the ends of microtubules. Interior to the edge, TRPC1 is found in diffuse areas around the microtubules (white arrow). Bars = 10 µm

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: Correlation coefficients for proteins colocalized with TRPC1 before and during SOCE

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: Loci containing STIM1, Orai, TRPC1, AQP4, and Vamp2. Results represent 3–8 experiments. a–f The peak radius for each vesicle size distribution in the untreated cell sample is indicated by an arrow and the same mark applied to samples collected during ER depletion and Ca 2+ readdition. a STIM1 peak at 240 nm, b TRPC1 peak at 200 nm, c cell surface TRPC1 peak at 215 nm, d AQP4 peak at 205 nm. e Vamp2 peak at 205 nm, f cells were fixed but not permeabilized after no treatment (untreated), after Ca 2+ depletion, or after Ca 2+ depletion followed by Ca 2+ readdition. TRPC1 shows a diffuse localization in cells 1 and 2. g Cumulative distribution of TRPC1 loci in samples of untreated cells and cells after ER depletion. The circumference within which loci are found near each filopodium is represented. Loci within 2.7 µm of the filopodia tip are slightly fewer during ER depletion. Statistics on the distances are untreated (mean = 3.27, S.E.M. 0.091 µm, 196 loci) and ER depletion (mean = 3.78, S.E.M. 0.13 µm, 187 loci). The number of particles segmented was closely related to the number of images (N) analyzed, and N was linear with the number of particles recovered within a single treatment (see Additional file : Fig. 6A–B)

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: Functional aquaporins and STIM1-labelled loci in relation to SOCE channels. a Areas with low refractive index are indicated by lower phase density. (Top) Low density areas are found immediately under the plasma membrane (arrowhead), in vesicles near the cell edge, in elevated portions of the cell, and in pointed protrusions (arrow). (Bottom) Linear structures with low density are present in the lamellipodium (bracket). b, c Confocal planes from cells after Ca 2+ readdition and colocalization with antibodies against STIM1 (blue), Orai (green), and TRPC1 (red). ( b , Left) Ventral plane with diffuse Orai (green) and numerous areas of STIM-TRPC colocalization in magenta (arrow). (Right) Dorsal plane of the same cells, c ventral plane of another area showing Orai colocalized with TRPC1 (yellow arrowhead) and areas of STIM1-TRPC1 colocalization (magenta loci). Inset: Nucleus of the same cell showing the diffuse distribution of Orai (green) and colocalization of all three proteins (white). Bars = 10 µm

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques: Functional Assay

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: Colocalization of PLC isoforms and TRPC1. a Triple localization of PLCβ2 (green), PLCγ1 (blue), and TRPC1 (red). PLCγ1 and TRPC1 coincide in interior parts of the cells (lavender). b, c Colocalization of PLCβ2 (green) and TRPC1 (red). b In an untreated cell, the labels coincide even at the cell edge (arrows). c After Ca 2+ readdition, TRPC1 is concentrated at the cell edge (bracket) but the two proteins are often colocalized at the interior of the cell (yellow). d, e Colocalization of PLCβ2 (red) and PLCγ1 (green). d In an untreated cell, the proteins coincide in many sites (yellow), but PLCβ2 can also occupy a separate compartment (arrowheads). e After Ca 2+ readdition, the proteins share compartments in some sites (yellow arrowheads) but not in others (green and red arrowheads). f Cumulative distribution of PLCβ2 particle sizes. The peak radius of loci in images from untreated cells is indicated. g, h Localization of PLCβ2 ( g ) and PLCγ1 ( h ) at the cell edge, showing a concentration at the pointed feature (arrowhead)

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques: Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

doi: 10.1186/s12964-022-00927-y

Figure Lengend Snippet: SOCE induces filopodia in the absence of an apparent Ca 2+ -binding structural element. a During ER depletion, TRPC1 (red) retreats to internal membrane compartments (middle frame). Although [Ca 2+ ] i is elevated, Ca 2+ flux through Orai is impossible because there is no extracellular Ca 2+ . Upon Ca 2+ addition, the TRPC-bearing vesicles undergo exocytosis. b Proposed mechanism of vesicle exocytosis. Vesicles are attached to microtubules and mobilized because of the Ca 2+ influx through Orai channels. The lack of Orai alignment in linear arrays (data not shown) suggests that Orai is not mobilized to the plasma membrane in the same way as TRPC1. c Putative mammalian signalplex underlies TRPC1 localization and dissemination. On the left side, TRPC1 (red), scaffold protein (brown), and ERM (gray and green) form an aggregate in the plasma membrane on a substrate of actin (blue). Upon Ca 2+ influx, TRPC channels dissociate from the scaffold (right side)

Article Snippet: Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 (R&D Systems, Minneapolis, MN), CaV1.2 (Novus Biologicals, Centennial, CO), caveolin-2 (Thermo Fisher, Rockford, IL), β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A (CRAC regulator 2A, Cedarlane Laboratories, Burlington NC), and a combination of five clones against PLCγ1 (Sigma-Aldrich).

Techniques: Binding Assay