Journal: The Journal of Physiology

Article Title: Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

doi: 10.1113/jphysiol.2012.240952

Figure Lengend Snippet: Representative immunofluorescence images of PLIN5 (A), IMTG (B), and the merged images (C) with the yellow areas describing regions of overlap between PLIN5 and IMTG images. The yellow objects were then extracted (to measure the number of PLIN5-LDs; D), and the number subtracted from the total number of LDs to quantify the number of PLIN5-null-LD. The same procedure was used to obtain values of colocalization between PLIN2 and IMTG. White bar, 5 μm. The number of PLIN2-LD (filled bars) and PLIN2-null-LD (open bars) were quantified before (PreEx) and after exercise (PostEx), prior to (E) and following (F) training in both the SIT and ET groups. The same analysis was repeated for PLIN5 for pre- (G) and post-training (H). *Main effect of exercise bout (P < 0.05 vs. pre-exercise).

Article Snippet: The specificity of the PLIN5 antibody was confirmed in a competition experiment in which a PLIN5 recombinant peptide (Cat. no. NB110–60511PEP; Novus Biologicals, Cambridge, UK) was incubated with the PLIN5 primary antibody, subsequently resulting in removal of the positive fluorescent signal for PLIN5 (Supplementary Fig. S1 ).

Techniques: Immunofluorescence

Journal: The Journal of Physiology

Article Title: Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

doi: 10.1113/jphysiol.2012.240952

Figure Lengend Snippet: Representative immunofluorescence images of PLIN5 (stained green) in combination with WGA to identify the cell border (stained blue) in skeletal muscle, pre- (A) and post- (B) 6 weeks of SIT. Type I fibres are indicated with a ‘I’, all other fibres are assumed type II fibres. White bar, 50 μm. PLIN5 expression, quantified from immunofluorescence images of PLIN5, in type I and type II fibres obtained before (C) and after (D) 6 weeks of SIT (filled bars) or ET (open bars). PLIN5 expression quantified from immunofluorescence images correlated with PLIN5 expression determined following immunoblotting of whole muscle homogenates (E). Values are presented as means ± SEM (n= 8 per group). *Main effect of fibre type (P < 0.05 vs. type I fibres). †Main effect of training intervention (P < 0.05 vs. pre-training).

Article Snippet: The specificity of the PLIN5 antibody was confirmed in a competition experiment in which a PLIN5 recombinant peptide (Cat. no. NB110–60511PEP; Novus Biologicals, Cambridge, UK) was incubated with the PLIN5 primary antibody, subsequently resulting in removal of the positive fluorescent signal for PLIN5 (Supplementary Fig. S1 ).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: The Journal of Physiology

Article Title: Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

doi: 10.1113/jphysiol.2012.240952

Figure Lengend Snippet: Bivariate correlation analysis

Article Snippet: The specificity of the PLIN5 antibody was confirmed in a competition experiment in which a PLIN5 recombinant peptide (Cat. no. NB110–60511PEP; Novus Biologicals, Cambridge, UK) was incubated with the PLIN5 primary antibody, subsequently resulting in removal of the positive fluorescent signal for PLIN5 (Supplementary Fig. S1 ).

Techniques:

Journal:

Article Title: Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes

doi: 10.1111/j.1469-7793.2001.0339c.xd

Figure Lengend Snippet: A, amplified DNA fragments of P2X receptors (lanes 1–7) from rat brain (a) and rat portal vein myocytes (b) were separated on a 2 % agarose gel and visualized by staining with ethidium bromide. Lane 8, RNA from brain and portal vein myocytes in the absence of reverse transcriptase served as a negative control. Numbers on the left indicate molecular size standards in base pairs (bp). For RNA purification and PCR conditions, see Methods. B, immunostaining of P2X receptor subtypes in portal vein myocytes. Myocytes were stained with anti-P2X1 receptor (a) or anti-P2X4 receptor antibody (b) and vizualization was realized with a donkey anti-rabbit IgG FITC-conjugated antibody. In the absence of primary antibodies or after inactivation of the antibodies by their antigen peptides, only a faint background fluorescence was observed (not shown). Typical confocal sections were performed above the nucleus and therefore appeared spherical. Both P2X1 (a) and P2X4 (b) receptor subtypes were distributed throughout the confocal sections with a marked staining of P2X1 receptor subtype at the cell periphery.

Article Snippet: The rabbit anti-P2X1 and anti-P2X4 receptor antibodies (Alomone Labs, Jerusalem, Israel) were directed against polypeptides corresponding to residues 382–399 and 370–388 of the rat P2X1 and P2X4 receptors, respectively.

Techniques: Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Purification, Immunostaining, Fluorescence

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

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: BAK antibody blocking peptide , ELISA detection , N/A , N/A , Novus Biologicals , NBP1-77152PEP , N/A.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay