trpc6 specific antiserum  (Alomone Labs)

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Schema of TRPC3/TRPC6 chimera. A, domains and motifs in the TRPC3 C terminus. B, representation of the proximal (C1) and distal (C2) C termini of TRPC3 and TRPC6. C, schematic models of TRPC3 chimeras: TRPC3-C6C1, TRPC3-C6C2, and TRPC3-C6TRP. D, schematic models of TRPC6 chimeras: TRPC6-C3C1, TRPC6-C3C2, TRPC6-C3TRP, and TRPC6-C3TRP-C3C2. For B–D, the origin of the TRP domain is shown by the color of the oval. The amino acid (AA) number at the beginning and at the end of the C1 and C2 domains contributed by TRPC3 (top) or TRPC6 (bottom) is indicated. FLAG-tagged chimeras expressed FLAG at the N terminus, and V5-tagged chimeras expressed V5 at the C terminus of the channel.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: TRP domains, leucine zipper motifs, and AMPK binding site in TRPC3 and TRPC6. A, amino acid compositions of TRP domains and leucine zipper motifs of TRPC3 and TRPC6 are shown. Letters in boldface type indicate exchanged amino acids in the chimeras. For TRP domain exchange, the boldface sequences in the TRP domain of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6TRP. The boldface amino acids of TRPC3 TRP were exchanged with those of TRPC6 to create TRPC6-C3TRP. For leucine zipper exchange, the boldface sequences in the leucine zipper of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6LZ. The boldface amino acids of TRPC3 were exchanged with those of TRPC6 leucine zipper to create TRPC6-C3LZ. B, exchange of TRPC3 741–748 and TRPC6 802–809 is shown. Amino acids contributed by TRPC3 (top) or TRPC6 (bottom) are indicated, and localization to the C1 or C2 part of the C terminus is shown.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Role of TRP domains in regulation of TRPC3 and TRPC6 by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC3-C6TRP, or BFP-TRPC6-C3TRP chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca2+]i, F440/F490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F440/F490 above base line (mean ± S.E. (error bars) percentage increase) = peak F440/F490 divided by base line F440/F490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 17, Epo 21), BFP-TRPC6 (PBS 17, Epo 22), BFP-TRPC3-C6TRP (PBS 16, Epo 22), or BFP-TRPC6-C3TRP (PBS 18, Epo 21). The Epo-stimulated increase in cells expressing TRPC6 and Epo-R is not statistically different from cells expressing Epo-R alone and is thought to be secondary to Epo-R activation of low levels of endogenous channels (17). *, significantly greater percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC6 (p < 0.001). **, significantly less percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC3 (p < 0.001).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Expressing, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Role of the distal C terminus of TRPC3 and TRPC6 in regulation by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3TRP, BFP-TRPC6-C3C2, BFP-TRPC6-C3TRP-C3C2, BFP-TRPC3-C6 802–809, or BFP-TRPC6-C3TRP-C3 741–748 chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca2+]i, F440/F490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F440/F490 above base line (mean ± S.E. (error bars) percentage increase) = peak F440/F490 divided by base line F440/F490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 56, Epo 102), BFP-TRPC6 (PBS 57, Epo 103), BFP-TRPC6-C3TRP (PBS 20, Epo 34), BFP-TRPC6-C3C2 (PBS 20, Epo 35), BFP-TRPC6-C3TRP-C3C2 (PBS 30, Epo 64), BFP-TRPC3-C6 802–809 (PBS 26, Epo 49), or BFP-TRPC6-C3TRP-C3 741–748 (PBS 12, Epo 30). *, significantly greater percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC6 (p < 0.001). **, significantly less percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC3 (p < 0.001).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Plasma membrane insertion of TRPC3/TRPC6 chimeras detected with cell surface biotinylation. HEK 293T cells transfected (Tx'd) with Epo-R and V5-TRPC3, V5-TRPC3-C6TRP, V5-TRPC6, V5-TRPC6-C3TRP, V5-TRPC6-C3C2, V5-TRPC6-C3TRP-C3C2, or V5-TRPC6-C3TRP-C3 741–748 were stimulated with 0–40 units/ml Epo for 0–5 min. Biotinylation of cell surface proteins was performed, and V5-tagged proteins were immunoprecipitated (IP) from lysates with anti-V5 antibody. Western blots (WB) of immunoprecipitates were probed with streptavidin-HRP to detect biotinylated protein and then stripped and reprobed with anti-V5-HRP to detect total protein. Representative results of Western blots from three experiments are shown. Biotinylated and total protein bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. (error bars) values of the biotinylated/total protein ratios from three experiments after 5 min of stimulation are shown. *, significant difference in the ratio compared with time 0 (p < 0.05).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A, proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting (WB). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B, representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na+K+-ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Purification, Western Blot, Construct, Fractionation

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells (A–C) or UT-7/Epo cells (C) were fractionated, purified, and analyzed by Western blotting (WB). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na+K+-ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A, fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B, fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C, subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys, whole cell lysate; M, membrane fraction; Csk, cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Purification, Western Blot, Construct, Fractionation, Marker, Cell Fractionation, Blocking Assay