Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 1. TRPC4 expression in adult mouse brain. A, B, In situ hybridization of TRPC4-mRNA in amygdala, hippocampus, somatosensory cortex, auditory thalamus, and auditory cortex. BLA, amygdala basolateral nucleus; S1, primary somatosensory cortex;S2,secondarysomatosensorycortex;AuT,auditorythalamus;AuD,secondaryauditorycortex,dorsal;Au1,primaryauditory cortex; AuV, secondary auditory cortex, ventral; Ect, ectorhinal cortex; PRh, perirhinal cortex. Scale bar, 1 mm. C, TRPC4 (left) and CaMKII(middle;amarkerofpyramidalneurons)colocalizeintheLA(right).D,CellsexpressingTRPC4(red)andGFAP(green;amarker ofglialcells)donotcolocalizeintheLA.E,CellsexpressingTRPC5(red)andGAD67(green;amarkerofinterneurons)donotcolocalizeinthe LA.Scalebars:C–E,10m.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Expressing, In Situ Hybridization

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure2. GenerationandconfirmationofTRPC4/mouse.A,TargetingstrategyforthedisruptionoftheTRPC4gene.Afterhomologousrecombination,deletionofexon4regionwascatalyzed by Cre-recombinase in ES cells. B, Targeting of the TRPC4 locus is confirmed by PCR analysis of tail genomic DNA. C, RT-PCR analysis of whole-brain mRNA from control and TRPC4/ littermates confirms the absence of exon 4. D, IP of TRPC4 protein in brain microsomes from control and TRPC4/ mice reveals loss of TRPC4 protein in TRPC4/ mice (top). Western blotting of NKA- confirmsequalproteinloadingincontrolandTRPC4/mice(bottom).E,ImmunohistochemicalstainingofbrainsectionsfromcontrolandTRPC4/littermatesrevealsselectivelossofTRPC4 expression in mutant mice. Scale bar, 1 mm.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 9. mGluR-EPSCs in LA neurons are suppressed in TRPC4/ mice. A, Synaptic responses in cortical input to the LA neuroninaslicefromacontrolmouseevokedbytrainsofhigh-frequencystimulationbeforeandduringadditionofCNQX(AMPA receptorantagonist;20M),NMDAreceptorantagonists,D-APV(50M),andMK-801(10m)andGABABRantagonistCGP35348 (300 M) recorded as described previously (Riccio et al., 2009). Stimulation trains consisted of 10 pulses at 100 Hz which were delivered once every 30 s. Inset shows synaptic responses recorded in current-clamp mode before (1) and after (2) the addition of antagonists to the external medium. The dashed line indicates the time point where the EPSP amplitude was measured. B, The experiment was identical to A, but the recording was obtained from a TRPC4/ mouse. C, Summary data for the experiments as in A and B, performed in both cortical and thalamic inputs to the LA. The amplitude of the residual component of the EPSP in the presence of antagonists was smaller in both pathways in slices from TRPC4/mice comparedwithcontrollittermates.D,EPSCsincorticalinputrecordedinvoltage-clampmodeatholdingpotentialsranging from –100 mV to 40 mV in slices from control (left) and TRPC4/ (right) mice in the presence of the antagonists (as in A). E, Current–voltage (I–V) plots of the peak current in cortical input (as in D) in slices from control and TRPC4/ mice. F, EPSCs in thalamic input recorded in voltage-clamp mode at holding potentials ranging from –100 mV to 40 mV in slices from control (left) and TRPC4/ (right) mice in the presence of the antagonists (as in A). G, Current–voltage (I–V) plots of the peak current in thalamic input (as in F) in slices from control and TRPC4/ mice. H, Left, Representative immunoblotshowssimilaramountsofTRPC5proteininbrainmicrosomesextractedfromcontrolorTRPC4/littermates. Western blotting of Na -K -ATPase indicates equal protein loading (bottom). Right, Quantification of three different blots. Results are shown as mean SEM.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Control, Western Blot

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 10. CCK-mediated increase in spike firing in LA neurons is diminished in TRPC4/ mice. A, Spikes evoked in LA neurons by current injection (150 pA) recorded in current-clamp mode under baseline conditions and in the presence of 3 M CCK4 in a slice from a control mouse. B, Summary plot showing CCK4-induced depolarization in LA neurons in slices from control and mutant mice. C, Spikes evoked in LA neuron under baseline conditions and in the presence of 3 M CCK4 in a slice from a TRPC4/ mouse. D, The percentage increase in spike frequency in the presence of CCK4 relative to the baseline frequency (taken as 100%) in slices from control and null mice. E, Left, Fluorescence double labeling for CCKergic fibers (green) and CaMKII (red) reveals no differences in innervation of the LA by CCK-containing fibers in brain sections from control and TRPC4/ littermates. Right, Mean CCK fluorescence after normalization to CaMKII fluorescence from six images per group taken from different stainings (two images from each of 3 control and 3 KO mice). Scale bar, 10 m. Results are shown as mean SEM.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Injection, Control, Mutagenesis, Fluorescence, Labeling

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized fold change in TRPC1/4/5 mRNA (2 –ΔΔ CT ) in TBI mice compared to sham.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Cell-type specific TRPC1, TRPC4, and TRPC5 channel upregulation in the hippocampus and cortex after CCI-TBI. (A,B) Representative Western immunoblots using (A) TRPC4 (∼120 kDa) and (B) TRPC5 (∼110 kDa) antibodies in sham and TBI cortex and hippocampus. Blots were normalized to β-actin protein (42 kDa) as loading control. (C,D) Summarized data for Western blot quantification of TRPC4 ( C , n = 7–13 animals per group) and TRPC5 ( D , n = 5–7 animals per group) from microdissected brain regions in mice 7 days after TBI. (E,F) Shown are summarized plots of percent difference in TRPC4 (E) and TRPC5 (F) protein between ipsilateral and contralateral hemispheres of microdissected regions from data in Panels (C,D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham of same subregion. † p < 0.05 vs. contralateral hemisphere of same subregion.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Western Blot, Control

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized TRPC4/TRPC5 protein in 7-day sham and TBI mice.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Surges in neuronal activity following CCI-TBI are TRPC4/TRPC5-mediated. (A) Representative images of parietal cortex from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. Prefix “ c ” denotes contralateral, prefix “ i ” denotes ipsilateral. Red = cFos-tdTomato; blue = DAPI. (B) Representative images of hippocampal subregions from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. (C) Summarized quantification of cFos+ neuron density (neurons/0.1 mm 3 ) in sham and TBI TRAP mice activated at the time of TBI, as in Panels (A,B) . (D) Representative images taken from sham mice, TBI mice, and TBI mice also administered M084 (10 mg/kg) (TBI + M084) that were administered 4-OHT t = 7 days after procedure. (E) Summarized quantification of cFos+ neurons in sham, TBI, and TBI + M084 mice 7 days after procedure, as in Panel (D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham. # p < 0.05 vs. TBI cDG. † p < 0.05 vs. TBI of same region. Scale bars: 100 μm.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activity Assay

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: TRPC4/TRPC5 channel activation artificially prolongs Ca 2+ influx in DGGCs after CCI-TBI. (A) Cumulative probability distribution of the peak amplitude of GCaMP6f fluorescence (ΔF/F) for each DGGC from sham (control) and iTBI slices during EA (1 μM, red) or EA + M084 (10 μM, gray) application. (B) Cumulative probability distribution of the Ca 2+ influx duration (in seconds) for each DGGC from control and iTBI slices during EA or EA + M084 application. (C,D) Histogram population distribution of control DGGC Ca 2+ influx events according to peak amplitude (C) and Ca 2+ event duration (D) . (E,F) Histogram population distribution of iTBI DGGC Ca 2+ influx events according to peak amplitude (E) and Ca 2+ event duration (F) . (G) Summarized means of peak fluorescence from data as in Panel (A) . (H) Summarized means of Ca 2+ influx duration from data as in Panel (B) . Red = EA alone, gray = EA + M084. All data bars represent the mean ± SEM. * p < 0.05 vs. EA alone from same procedure condition, † p < 0.05 vs. control of same drug condition.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activation Assay, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE 1. hTRPC4 is tyrosine-phosphorylated following EGF stimulation. A, Alomone anti-TRPC4 antibody recognizes transfected hTRPC4 and an endogenous species of TRPC4. COS-7 cells were transfected with FLAG-hTRPC4 or YFP-FLAG-hTRPC4, and 20 g of total protein was immunoblotted with anti-TRPC4 antibody, then stripped and re-probed with anti-FLAG antibody. B, EGF receptor stimulation promotes rapid tyrosine phosphorylation of hTRPC4. COS-7 cells transiently transfected with FLAG-hTRPC4 were stimulated with EGF (100 ng/ml, 20 min), reacted with anti-FLAG antibody, and immunoprecipitated hTRPC4 was blotted for phosphorylated tyrosine residues with anti-PY20 (top) or anti-FLAG M2 (bottom) antibodies. Precipitation from untransfected COS-7 cells was used as a negative control. Shown also is whole cell lysate (7% of immunoprecipitation input). Lower panel, phosphorylated tyrosine levels were determined using ImageQuant software and expressed as a percentage of unstimulated levels and normalized for total protein content (***, p 0.001 compared with control). C, YFP-hTRPC4 is tyrosine-phosphorylated in response to EGF. COS-7 cells transfected with YFP-hTRPC4 stimulated with EGF (100 ng/ml, 20 min). YFP-hTRPC4 precipitated with anti-GFP antibody was blotted with anti-PY20 (top) and anti-TRPC4 (bottom) antibodies. D, endogenous hTRPC4 is tyrosine-phosphorylated following EGF treatment. A431 cells were stimulated with EGF (50 ng/ml, 20 min) in the presence or absence of AG1478 (10 nM) followed by precipitation with anti-PY20 (PY20) antibody. Bound proteins were analyzed by blotting with anti-TRPC4 antibody. E, TRPC1 does not undergo tyrosine phosphorylation following EGFR stimulation. COS-7 cells transfected with GFP-hTRPC1 were stimulated with EGF (100 ng/ml, 20 min) and reacted with anti-GFP antibody, and bound proteins were analyzed by blotting with anti-PY20 (top) or anti-GFP (bottom)antibodies.F,onlyEGFRstimulationinducedsignificanttyrosinephosphorylationofhTRPC4.FLAG-TRPC4transfectedCOS-7cellsweretreatedwithEGF(100ng/ml,15min), carbachol (CCh, 10 M, 10 min), oleoylacyl-sn-glycerol (OAG, 50 M, 10 min), histamine (HIS, 100 M, 10 min), or thapsigargin (Tg, 1 M, 10 min), and immunoprecipitated hTRPC4 was assessed for tyrosine phosphorylation with anti-PY20 antibody (top). Total protein was determined with anti-TRPC4 antibody (middle). Whole cell lysates separated by SDS-PAGE and analyzed by blotting with anti-phosphorylated ERK1/2 or anti-PY20 (bottom) antibodies are shown for comparison of global phosphorylation levels. Phosphorylated tyrosine levels were determined using ImageQuant software and expressed as a percentage of unstimulated levels and normalized for total protein content (*, p 0.05 compared with control).

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Phospho-proteomics, Immunoprecipitation, Negative Control, Software, Control, SDS Page, Comparison

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE 2. Src family tyrosine kinases (STKs) mediate hTRPC4 tyrosine phosphorylation. A, PP2 inhibits EGF-mediated tyrosine phosphorylation of hTRPC4. COS-7 cells trans- fected with FLAG-hTRPC4 were preincubated with either PP2 (5 M) or PP3 (5 M) for 10 min prior to EGF (100 ng/ml, 20 min) application. FLAG-hTRPC4 precipitated with anti-FLAG antibody was blotted with anti-PY20 antibody (top). Total protein levels were determined by blotting with anti-TRPC4 antibody (bottom). Phosphorylated tyrosine levels were determined using ImageQuant software and expressed as a percentage of unstimulated control levels and normalized for total protein content (**, p 0.01 compared with control). EGFR was also precipitated from COS-7 cells stimulated with EGF in the presence of PP2 and analyzed for phosphorylated tyrosine levels (middle panel). The bottom panel shows precipitated EGFR immunoblotted for anti-PY20. B, Fyn promotes hTRPC4 tyrosine phosphorylation. COS-7 cells were transfected with FLAG-hTRPC4 and either Fyn-KM, Fyn-WT, or Fyn-YF, and at 48 h post-transfection hTRPC4 was precipitated with anti-FLAG antibody and blotted with anti-PY20 (top) or anti-TRPC4 (bottom) antibodies. Phosphorylated tyrosine levelsweredeterminedusingImageQuantsoftwareandexpressedasapercentageofunstimulatedcontrollevelsandnormalizedfortotalproteincontent(**,p0.01;***,p0.001 compared with control). C, expression of Fyn-KM prevents EGF-induced phosphorylation of hTRPC4. COS-7 cells transfected with FLAG-hTRPC4 and either control vector or Fyn-KM were stimulated at 48 h post-transfection with EGF (100 ng/ml, 20 min), cell lysates reacted with anti-FLAG antibody and precipitates analyzed by blotting with anti-PY20 (top) or anti-TRPC4 (middle) antibodies. Phosphotyrosine immunoblotting of immunoprecipitated EGFR from COS-7 cells transfected with Fyn constructs is shown below (n 7). D, Lyn also interacts with and promotes tyrosine phosphorylation of hTRPC4. COS-7 cells transfected with Lyn-WT or p53 and p56 Lyn-YF together with FLAG-hTRPC4 were lysed, reacted with anti-FLAG antibody, and blotted with anti-PY20 (top), anti-TRPC4 (middle), and anti-Lyn (bottom) antibodies. E, kinase-inactive Lyn (Lyn-Y397F/ki) does not prevent hTRPC4 phos- phorylation.COS-7cellsco-transfectedwithLyn-kiandhTRPC4werestimulatedwithEGF,precipitatedwithanti-FLAG,andblottedforphosphotyrosineandTRPC4.Thebottompanel shows collated data analyzed from seven individual experiments with ImageQuant.

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Software, Control, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Construct

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE 3. Fyn, Src, and Lyn associate with TRPC4. A, Fyn co-immunoprecipitates hTRPC4 from COS-7 cells. Cells were co-transfected with Fyn-WT and hTRPC4, immunoprecipitated with anti-Fyn antibody, and blotted with PY20, anti- FLAG, and anti-Fyn antibodies. B, Src associates with hTRPC4 in COS-7 cells. EGF-stimulated FLAG- hTRPC4-transfected COS-7 cells were immunopre- cipitated with anti-Src antibody, and precipitates were probed for the presence of phosphotyrosine, TRPC4, and Src. C, Lyn and Fyn physically associate with native TRPC4 in COS-7 cells. COS-7 cell lysates were immunoprecipitated with indicated anti- bodies and immunoblotted for TRPC4 (top), Lyn (middle), and EGFR (bottom).

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE 4. The C terminus of hTRPC4 is dually phosphorylated on tyrosine residues. A, Fyn phosphorylates tyrosine residues in the C termi- nus of TRPC4 in vitro. GST fusion proteins of the N and C termini of hTRPC4 were purified and sub- jected to an in vitro kinase assay with immunopre- cipitated Fyn-YF. Reactions were analyzed by blot- ting with anti-PY20 antibody. Anti-GST antibody was used to indicate total protein concentration (bottom panel). B, the distal 131 amino acids of hTRPC4 are critical for Fyn-mediated tyrosine phosphorylation of hTRPC4. COS-7 cells were transfected with either truncated C-terminal GFP- hTRPC4 (GFP-CT-T, amino acids 846–977) alone or together with the constructs indicated, reacted with anti-GFP antibody, and bound protein ana- lyzed for phosphotyrosine activity using anti-PY20 antibody (top). The second lane is from COS-7 cells treated with EGF (100 ng/ml, 20 min). Anti-GFP antibody was used to confirm equal fusion protein levels (bottom). C, Tyr-959 and Tyr-972 are tyro- sine-phosphorylated in response to EGF stimula- tion. Point mutations were introduced into FLAG- hTRPC4 (Y959F, Y972F, and Y959F/Y972F) and FLAG-hTRPC4 and mutated constructs transfected into COS-7 cells. Cells were stimulated with EGF (100 ng/ml, 20 min), and precipitated hTRPC4 was analyzed by blotting with anti-PY20 (top) and anti- TRPC4 (bottom) antibodies. Phosphotyrosine lev- els were quantified with ImageQuant software and corrected for total hTRPC4 content in immu- noprecipitates. Collated data are expressed as a percentage of control unstimulated wild-type (WT) hTRPC4 levels.

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: In Vitro, Purification, Kinase Assay, Protein Concentration, Phospho-proteomics, Transfection, Construct, Activity Assay, Software, Control

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE 5. EGF stimulates the translocation of TRPC4 to the plasma membrane. A, EGFR activa- tion induces the rapid insertion of hTRPC4 into the plasma membrane. COS-7 cells transfected with FLAG-hTRPC4 stimulated with EGF (100 ng/ml, 20 min) and cell surface proteins biotinylated, precip- itated with anti-FLAG antibody, analyzed by SDS- PAGE, and visualized with streptavidin-AP (top) and anti-TRPC4 antibody (bottom). TRPC4 plasma membrane levels were determined using Image- Quant software and expressed as a percentage of un-stimulated control levels and normalized for total protein content. B, tyrosine phosphorylation of hTRPC4 accompanies its membrane insertion. COS-7 cells were stimulated in parallel for the indi- cated times with EGF (100 ng/ml) and membrane proteins biotinylated, precipitated with anti-FLAG antibody and blotted in parallel for either biotiny- lated hTRPC4 with streptavidin-HRP (left panel) or tyrosine-phosphorylated hTRPC4 with anti-PY20 antibody (right panel). C, plasma membrane expression of endogenous hTRPC4 is increased by EGF application. A431 cells were stimulated with EGF (50 ng/ml, 20 min) and biotinylated, and lysates were incubated with NeutrAvidin-Sepha- rose. Bound proteins were analyzed by blotting with indicated antibodies (upper panel, TRPC4; lower panel, 4 integrin). hTRPC4 plasma mem- brane levels were determined using ImageQuant software and expressed as a percentage of unstimulated control levels and normalized for total protein content. D, FLAG-hTRPC4 membrane insertion is independent of the actin cytoskeleton. COS-7 cells transfected with FLAG-hTRPC4 were pre-treated with 10 M cytochalasin D for 20 min prior to stimulation with EGF (100 ng/ml). Cells were biotinylated, and hTRPC4 was precipitated with anti-FLAG antibody followed by blotting in parallel with anti-PY20 followed by anti-TRPC4 antibodies and with streptavidin-HRP conjugate. The control lane has been shifted to remove non- applicable lanes. Collated data quantified with ImageQuant are shown below.

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Transfection, SDS Page, Software, Control, Phospho-proteomics, Expressing, Incubation

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE 6. Tyrosine phosphorylation promotes plasma membrane insertion of hTRPC4. A, pre- incubation of COS-7 cells with PP2 prevents EGF- induced hTRPC4 translocation. FLAG-hTRPC4- transfected COS-7 cells stimulated with EGF (100 ng/ml,20min)eitherinthepresenceorabsenceof 5 M PP2 were biotinylated, cell lysates were reacted with anti-FLAG antibody, and precipitates were blotted with streptavidin-AP followed by anti-TRPC4 antibody. Biotinylated proteins were analyzed with ImageQuant and compiled in Prism. B, co-expression of Fyn-YF increases the plasma membrane levels of hTRPC4 in the absence of EGF stimulation. COS-7 cells were transfected with FLAG-hTRPC4 together with either control plas- mid, Fyn-YF, or Fyn-KM and biotinylated, and lysates were incubated with anti-FLAG antibody. Biotinylated hTRPC4 was visualized with streptavi- din-AP followed by anti-TRPC4 antibody. Protein levels were analyzed with ImageQuant, normal- ized for total hTRPC4 protein, and compiled in Prism. C, Fyn-KM prevents EGF-stimulated hTRPC4 membrane insertion. COS-7 cells transfected with indicated constructs were stimulated with EGF, and lysates were precipitated with anti-FLAG anti- body. Bound proteins were analyzed by Western blotting for biotin incorporation (upper) and total TRPC4 content (lower). Below are compiled data from five experiments.

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Clinical Proteomics, Membrane, Incubation, Translocation Assay, Transfection, Expressing, Control, Construct, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

doi: 10.1074/jbc.m503646200

Figure Lengend Snippet: FIGURE7.TyrosinephosphorylationenhancestheassociationofhTRPC4withNHERF.A,EGFstimulationenhancestheassociationofhTRPC4withNHERF.COS-7cellstransiently expressing HA-NHERF and FLAG-hTRPC4 were stimulated with EGF (100 ng/ml, 20 min), and cell lysates were reacted with anti-FLAG antibody. Bound proteins were analyzed for phosphorylated tyrosine levels (top), total hTRPC4 (middle), and NHERF (lower) content. B, EGF-stimulated NHERF association is inhibited by PP2. COS-7 cells transfected with FLAG-hTRPC4 and HA-NHERF were stimulated with EGF (100 ng/ml) in either the presence or absence of PP2 (5 M) and biotinylated, and lysates were reacted with anti-FLAG antibody. Western blots were performed as indicated. C, NHERF preferentially associates with tyrosine-phosphorylated hTRPC4. COS-7 cells transfected with HA-NHERF and FLAG-hTRPC4 were stimulated with EGF (100 ng/ml for 15 min), and lysates were immunoprecipitated with either anti-NHERF or anti-FLAG antibodies. Bound proteins were immunoblotted with anti-phosphotyrosine antibody (PY20, upper panel), then stripped and re-probed with anti-TRPC4 antibody (middle panel). Also shown is an immunoblot of precipitated NHERF. Data were collated and expressed as a percentage of either phosphotyrosine or anti-TRPC4 immunoreactivity relative to FLAG immunoprecipitates. D, EGF induces the movement of hTRPC4 to the cell membrane. HA-NHERF- and YFP-hTRPC4-transfected COS-7 cells were treated with or without EGF (100 ng/ml, 20 min), fixed, stained with anti-HA antibody, and visualized with streptavidin-Alexa594 tertiary. E, C-terminal tyrosine residues influence the binding of hTRPC4 to NHERF. Wild-type and mutated hTRPC4 constructs (WT, Y959F, Y972F, or Y959F/Y972F) transfected into COS-7 cells were precipitated with anti-FLAG antibody and assessed for NHERF (bottom) and tyrosyl (top) immuno- reactivity following EGF stimulation (100 ng/ml, 20 min). NHERF levels were quantified and expressed as a percentage of binding to unstimulated WT-hTRPC4. F, Y959F/Y972F mutant fails to translocate in response to EGF stimulation. COS-7 cells transfected with either wild-type hTRPC4 or dual mutant (Y959F/Y972F) were treated with EGF, biotinylated, and assessed for membrane insertion. Below are collated data from 10 experiments.

Article Snippet: Anti-FYN (FYN3), anti-Lyn, anti-EGFR, anti-Src, anti- 4 integrin, and anti-TRPC4 (sc-20) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Membrane, Staining, Binding Assay, Construct, Mutagenesis

Journal: Andrology

Article Title: Increased expression of TRPC4 channels associated with erectile dysfunction in diabetes.

doi: 10.1111/j.2047-2927.2014.00214.x

Figure Lengend Snippet: Figure 1 RT-PCR analysis of TRPC mRNA in cor- poral smooth muscle of human (A; a: cell and b: tissue) and rat (B). TRPC1, TRPC3, TRPC4 and TRPC6 mRNA were expressed in human penile tissues, cultured human corpus smooth muscle (CSM) cells and rat CSM tissues, whereas the mRNA of the TRPC5 and TRPC7 isoforms were not. Glyceraldehyde 3-phosphate dehydrogen- ase (GAPDH) served as a loading control and RNA input internal control.

Article Snippet: Specific antibody were used with a mouse monoclonal antiTRPC1 antibody (1 : 2000 dilution; Santa Cruz, CA, USA), a mouse monoclonal anti-TRPC4 antibody (1 : 2000; Neuromab, Davis, CA, USA), a rabbit polyclonal anti-TRPC6 antibody (1 : 500; Alomone Laboratories, Jerusalem, Israel) or a monoclonal anti-b-actin antibody (1 : 5000; Abcam, Cambridge, MA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Control