Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in dormant blastocysts via CB1. (A) Localization of CB1 in dormant and activated blastocysts. The trophectoderm cell surface is decorated with CB1. The levels of CB1 are significantly lower in activated blastocysts than those in dormancy. (B) Rapid activation of ERK by anandamide (ANA) in blastocysts. Dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide for the indicated times in minutes. Increased phosphorylation of ERK1/2(p-ERK1/2) and its translocation into nuclei were observed in dormant blastocyst trophectoderm cells within 5 min of their exposure to 7 nM anandamide, reaching a peak between 15 and 30 min. (C) Activation of ERK by anandamide is dose-dependent. Dormant blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide for 15 min. Anandamide at 7 nM activated ERK1/2 in dormant blastocyst trophectoderm cells, whereas it failed to do so at 28 nM. A CB1-selective antagonist SR141716A (SR1) at 7 nM or a MEK1/2 inhibitor U0126 at 1 μM inhibited the activation of ERK1/2 by 7 nM anandamide. (D) Total ERK remained unchanged. No changes in immunointensity for total ERK1/2 were observed in dormant blastocysts exposed to 7 nM anandamide or the vehicle. (E–G) Differential activation of ERK signaling by anandamide in CB mutant dormant blastocysts. CB1–/–, CB2–/–,or CB1–/– × CB2–/– dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide. Activation of ERK1/2 in the presence of 7 nM anandamide for 15 min was abrogated by the CB1 antagonist SR141716A (SR1) in CB2–/– blastocyst, but not in CB1–/– or CB1–/– × CB2–/– blastocysts. Images shown depict TRITC-labeled antigens in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bars, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Translocation Assay, Mutagenesis, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in normal day-4 blastocysts via CB1. Day-4 blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide (ANA) for 15 min. Activation of ERK1/2 at lower (7 nM) but not higher (28 nM) anandamide concentration was observed. A CB1-selective antagonist SR141716A (SR1) inhibited the accumulation of phospho-ERK1/2 (p-ERK1/2) by 7 nM anandamide. Images depict TRITC-labeled antigen in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bar, 20 μm.) Tr, trophectoderm; ICM, inner cell mass.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Concentration Assay, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Cannabinoid agonist CP55,940 induces activation of ERK in differentiating TS cells via CB1. (A) CB1 is expressed in TS cells. This cell line is stably transfected with the GFP gene. Images depict GFP in green, CB1 in red (TS cell surfaces), and the merge in yellow. (Scale bar, 50 μm.) (B and C) Activation of ERK in TS cells by CP55,940 (CP). TS cells were plated and expanded for 48 h. The cells were serum-starved for 5 h then exposed to different concentrations of CP for 15 min or 7 nM CP for the indicated times or to CP at 7 nM in the presence or absence of a MEK1/2 inhibitor (U0126), CB1-selective antagonist SR141716A (SR1), or CB2-selective antagonist SR144528 (SR2) for 5 min. Phosphorylation of ERK1 in differentiating TS cells was rapidly induced by 7 nM CP. U0126 or SR1, but not SR2, inhibited this activation. Quantitative analysis of ERK activation in C is expressed as percentage relative to the maximum band intensity.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Stable Transfection, Transfection

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Inhibition of depolarization-induced Ca2+ influx by 28 nM anandamide in dormant blastocysts. (A) Distribution of Ca2+ channel α subunits, α1B (N-type) and α1C (L-type), in dormant blastocysts. Both inner cell mass (ICM) and trophectoderm cell (Tr) are decorated with α1B and α1C subunits shown in red, Hoechst-labeled nuclei in blue, and the merge in pink. (B) Inhibition of depolarization-induced Ca2+ influx by anandamide (ANA). Ca2+ mobilization in blastocysts was visualized with Fluo-4 acetoxymethyl ester. Depolarization-induced Ca2+ influx in dormant blastocysts after exposure to 60 mM KCl was dramatically inhibited by 28 nM anandamide but not by 7 nM. The CB1-selective antagonist SR141716A (SR1) at equimolar concentration reversed this inhibition, but the CB2-selective antagonist SR144528 (SR2) was ineffective. The relative level of intracellular Ca2+ is indicated by the fluorescent intensity, which is displayed in pseudocolor according to the color bar by using lsmib. (Scale bar, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Inhibition, Labeling, Concentration Assay

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Anandamide at 7 nM confers blastocyst competency to implantation via CB1

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques:

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) Schematic of rabbit cardiac α 1C and β subunits. Red dots indicate putative PKA phosphorylation sites. (b) Schematic of binary transgene system. The expression of reverse tetracycline-controlled transactivator (rtTA) is driven by the cardiac-specific α-myosin heavy chain promoter. The cDNAs for FLAG-DHP-resistant (DHP*) α 1C or GFP-β 2B were ligated behind 7 tandem tetO sequences. (c) Exemplar whole-cell Ca V 1.2 currents of 35-mutant α 1C transgenic mice cardiomyocytes in nisoldipine before (black trace) and after isoproterenol (blue trace). Representative of 25 experiments. (d) Fold-change of peak DHP-resistant Ca 2+ current at 0 mV caused by isoproterenol or forskolin. Mean ± SEM. P =0.39 by unpaired two-tailed t-test. n= 45 cardiomyocytes from 5 mice, n = 25 cardiomyocytes from 5 mice. (e-f) Exemplar whole-cell Ca V 1.2 currents of GFP-tagged-28-mutant β 2B transgenic mice cardiomyocytes, and 35-mutant α 1C X 28-mutant β 2B transgenic mice cardiomyocytes. Representative of 8 and 22 independent experiments respectively. (g) Fold-change in peak Ca 2+ current caused by isoproterenol or forskolin for cardiomyocytes isolated from transgenic mice expressing GFP-tagged WT β 2B subunit , GFP-tagged 28-mutant β 2B , or both 35-mutant α 1C and GFP-tagged 28-mutant β 2B . Mean ± SEM. P =0.27 by one way-ANOVA. n= 19, 8, 21 cardiomyocytes from 4, 4, 3 mice, from left to right.

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Transgenic Assay, Two Tailed Test, Isolation

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) The 35 putative PKA phosphorylation sites in rabbit α 1C . The 51 residues in red are either predicted phosphorylation sites or within the immediate region of the predicted phosphorylation sites. All 51 residues were replaced with Ala in the 35-mutant α 1C transgenic mice. (b) Combined bar and column scatter plot of Boltzmann function parameters V 50 . Mean ± SEM. ** P <0.01; *** P <0.001; **** P <0.0001 by paired two-tailed t-test. pWT α, n= 19; 35-α mutant, n= 14; 28-β mutant, n= 16; 35-α mutant X 28-β mutant, n= 24. Specific P values can be found in the Source Data associated with this figure. (c) Graph of isoproterenol and forskolin-induced increase in nisoldipine-resistant current stratified by total basal current density before nisoldipine. (d) The 28 putative PKA phosphorylation sites in the N-terminal (NT), Hook, GK and C-terminal (CT) domains of β 2B . The 37 residues in red are either predicted phosphorylation sites or within the immediate vicinity of predicted phosphorylation sites, and were mutated to Ala in the 28-mutant GFP-tagged β 2B transgenic mice. (e) Fluorescence imaging of isolated cardiomyocytes expressing GFP-tagged 28-β mutant. Representative of images from more than 5 biologically independent mice. (f) Anti-β antibody immunoblot of cleared lysates from doxycycline-fed 35-mutant α 1C transgenic mice or 35-mutant α 1C X GFP-tagged 28-mutant β 2B expressing mice hearts. Representative of immunoblots obtained from at least 3 biologically independent mice. (g) Anti-FLAG antibody (upper) and anti-β antibody (lower) immunoblots of anti-FLAG antibody immunoprecipitations from cleared lysates of hearts from pWT, 35-α and three 35-α X GFP-tagged-28-β expressing mice. Representative images of two independent experiments. For source gel data, see .

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Phospho-proteomics, Mutagenesis, Transgenic Assay, Two Tailed Test, Fluorescence, Imaging, Isolation, Expressing, Western Blot

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) Exemplar current-voltage relationship of Ca 2+ currents from α 1C -APEX2 mice cardiomyocytes acquired in absence (black trace) and presence of nisoldipine (red trace). Inset scale bars: horizontal 100 ms, vertical 10 pA/pF. Representative of 5 experiments. (b) Time course of changes in sarcomere length after superfusion of nisoldipine-containing solution. Representative of 7 experiments (c) Percent-shortening in the presence of nisoldipine. Mean ± SEM. ****, P <0.0001 by unpaired two-tailed t-test. n=12, 7 cardiomyocytes, respectively. (d) Immunofluorescence of cardiomyocytes isolated from α 1C -APEX2 and β 2B -APEX2 expressing mice exposed to biotin-phenol and H 2 O 2 or no H 2 O 2 . Nuclear labeling with DAPI stain. Scale bar = 5 μm. Representative of 5 and 8 cardiomyocytes from 2 and 3 mice respectively. (e) Streptavidin-horseradish peroxidase (HRP) blot of lysates of isolated ventricular cardiomyocytes. Blot representative of 5 similar experiments. (f) Exemplar whole-cell Ca V 1.2 currents recorded from cardiomyocytes of α 1C -APEX2 transgenic mice. Black trace: 300 nM nisoldipine; blue trace: 200 nM isoproterenol + nisoldipine. Representative of 9 cells from 2 biologically independent mice. (g) Same as (f) except from β 2B -APEX2 transgenic mice. Black trace: control; blue trace: 200 nM isoproterenol. Representative of 7 experiments from 2 biologically independent mice. (h-j) Anti-phospho-phospholamban antibody immunoblot of isolated cardiomyocytes exposed to 1 μM isoproterenol. For cardiomyocytes isolated from α 1C -APEX2 and β 2B -APEX2 mice, the cardiomyocytes were exposed to isoproterenol after incubation with biotin-phenol. Blots representative of 3 independent experiments from at least 5 biologically independent mice for each genotype. (k) Same as (h-j) except whole heart exposed to 1 μM isoproterenol for 5 minutes after infusion of biotin-phenol. Blots representative of at least 5 biologically independent mice for no isoproterenol and at least 5 biologically independent mice for isoproterenol. For source gel data, see .

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Two Tailed Test, Immunofluorescence, Isolation, Expressing, Labeling, Staining, Transgenic Assay, Control, Western Blot, Incubation

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) Immunofluorescence of isolated α 1C -APEX2 and β 2B -APEX2 cardiomyocytes exposed to biotin-phenol and H 2 O 2 . Representative of 13 and 8 cardiomyocytes from 2 and 3 mice respectively. Scale bar = 5 μm. (b) Immunofluorescence of tissue sections of Langendorff-perfused α 1C -APEX2 heart. Scale bars: upper −100 μm; lower − 5 μm; lower inset− 5 μm. Representative of 10 sections from 2 mice. (c) Immunoblots of biotin-labeled proteins from α 1C -APEX2 and β 2B -APEX2 mice cardiomyocytes. In contrast to Ca V 1.2 subunits, RyR2, Jph2 and CaM, K V 1.5 channels were not detected in streptavidin-pulldown. Blots representative of 3 independent experiments. (d) Schematic of workflow for isolated cardiomyocytes. (e) Volcano plot of fold-change for relative protein quantification by TMT mass spectrometry of α 1C -APEX2 samples. Data shown are means for 5 pairs of biologically-independent samples. Non-adjusted unpaired two-tailed t-test. Rad (red dot) is reduced by 50% and PKA catalytic subunit (green dot) is increased by 50%. (f) Same as (e) except cardiomyocytes from β 2B -APEX2 mice. Data shown are means for 3 pairs of biologically independent samples. Rad is reduced by 30% and PKA catalytic subunit is increased by 68%. (g) Schematic of protein labeling and workflow for Langendorff-perfused α 1C -APEX2 mice hearts. bpm= beats per minute. (h) Same as (d) except proteins from α 1C -APEX2 whole heart samples. Data shown are means for 10 hearts, 5 without isoproterenol and 5 with isoproterenol. Rad is reduced by 36% (i) Schematic of workflow for isolated cardiomyocytes from non-transgenic (NTG) mice. (j) Same as (e) except proteins isolated from non-transgenic (NTG) mice cardiomyocytes without biotinylation or pull-down. * single peptide ESFDSQSLINNQSK. Data shown are means for 4 pairs of biologically-independent samples. Rad (red dot) in whole heart is unchanged by isoproterenol. For source gel data, see .

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Immunofluorescence, Isolation, Western Blot, Labeling, Mass Spectrometry, Two Tailed Test, Transgenic Assay

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) Proteins with a ratio of >2 (measured by normalized TMT signal/noise) in the experimental conditions compared to a no-labeling control (no H 2 O 2 ) are sorted by spectral counts. The 150 proteins with the highest peptide counts are displayed in the color-coded table. α 1C -APEX2 and β 2B -APEX2 data were collected in biological duplicate experiments. The full table with 3883 proteins quantified by multiplexed SPS MS 3 TMT mass spectrometry is reported in . (b) Prefuse force directed map of proteins from (a). Peptide counts were used as weight. Proteins mapping to the GO-terms “Z disc” are colored in green, to “membrane” in yellow and to both in purple. The α 1C -APEX2 and β 2B -APEX2 are colored in blue. (c) Gene-ontology (GO) term (cellular localization) enrichment for proteins in (a). The full table is reported in .

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Labeling, Control, Mass Spectrometry, Membrane

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) Dendrogram showing two-way hierarchical clustering of scaled TMT signal to noise (s/n) data for streptavidin-purified proteins from α 1C -APEX2 expressing cardiomyocytes after vehicle or after isoproterenol stimulation. Scaled relative TMT protein quantification data for 1951 proteins from biological quintuplicate α 1C -APEX2. Clustering used Ward’s minimum variance method. (b) Dendrogram showing two-way hierarchical clustering of scaled relative quantification data for 1936 proteins from biological triplicate β 2B -APEX2 experiments. Heterogeneity between cardiomyocyte preparations from different mice is apparent. (c) Dendrogram showing two-way hierarchical clustering of scaled relative quantification data for 2610 proteins from whole-organ α 1C -APEX without or with perfusion of isoproterenol. Prominent heterogeneity in relative protein quantification between hearts is apparent. Position of Rad is indicated by a red line. In this experiment, the individual hearts are not paired. (d) Dendrogram showing two-way hierarchical clustering of scaled TMT signal to noise (s/n) data from non-transgenic (NTG) cardiomyocytes under isoproterenol stimulation or with vehicle. Scaled data of 4622 quantified proteins from biological quadruplicate experiment are displayed. Pairing of samples is apparent.

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Purification, Expressing, Quantitative Proteomics, Transgenic Assay

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) MS 2 spectrum and TMT quantification parameters of a Rad peptide changed upon isoproterenol treatment of murine hearts. The MS 2 spectrum used for identification of the peptide: IFGGIEDGPEAEAAGHTYDR mapping to Rad is displayed. y and b ion m/z identified in the spectrum and their deviation from theoretical m/z are displayed on the left. Precursor mass was measured as 778.71 Da with a charge of +3. Peptide modifications were +229.16 Da for TMT on the peptide N-terminus and lysine residues, +57.02 Da for cysteine alkylation and +15.99 for methionine oxidation. Ion injection times, isolation specificity, sum of signal to noise (SN) over all TMT channels, TMT raw intensities, adjusted intensities and final SN intensities used for relative quantification as well as SPS ion m/z are displayed. (b) Table showing gene names of proteins with P < 0.05 for the three approaches: cardiomyocytes isolated from α 1C -APEX and β 2B -APEX mice, and α 1C -APEX hearts. Yellow-highlighted genes are in common amongst groups, but note for Mast2, the fold-change is not consistent. D3Z0N2* is a single peptide with the sequence ESFDSQSLINNQSK and its abundance is reduced by isoproterenol-treatment in non-transgenic cardiomyocytes to 27% likely by post-translational modification (P =0.000002, see and ). Data are mean fold-change for 5 pairs of biologically independent pairs of α 1C -APEX2 cardiomyocyte samples, 3 pairs of biologically independent pairs of β 2B -APEX cardiomyocyte samples, and 10 α 1C -APEX2 hearts, 5 without isoproterenol and 5 with isoproterenol. Non-adjusted unpaired two-tailed t-test. (c) Venn diagram of the data from (b). Proteins: Rad = Rrad; PKA catalytic subunit= Prkaca; Acss1 = acyl-CoA synthetase short chain family member 1. Rad is the only protein consistently changed amongst the three approaches.

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Injection, Isolation, Quantitative Proteomics, Sequencing, Transgenic Assay, Modification, Two Tailed Test

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a, c, e, f) Ba 2+ current elicited by voltage ramp every 10s. Black traces before and blue traces after forskolin. Representative of 15,16, 8 and 13 cells, respectively. (b, d) Diary plot of normalized Ba 2+ current amplitude at 0 mV. Representative of 15 and 16 cells. (g) Fold-change in maximum conductance (G max ) induced by forskolin. Mean ± SEM. P < 0.0001 by one-way ANOVA; ** P < 0.01, **** P < 0.0001 by Tukey’s test. n= 27, 76, 9, 23, 18 cells, from left to right. Specific P values can be found in the Source Data associated with this figure. (h) Boltzmann function parameter V 50 . Mean ± SEM. *** P <0.001 by two-tailed paired t-test. n= 15, 16, 8, 13, from left to right. ( i) Fold-change in G max induced by forskolin in absence and presence of Rad. Mean ± SEM. **** P < 0.0001 by unpaired two-tailed t-test. n= 7, 16, left to right. (j-l) The top rows display stochastic records, where channel closures are zero-current portions of the trace (horizontal gray lines) and openings are downward deflections to the open level (slanted gray lines). Bottom row: pale blue and gray lines are average P O -V relationship from multiple cells. Blue and black lines are Boltzmann fits. In all experiments, α 1C and β 2B were expressed in HEK cells with no Rad (j), WT Rad (k), or 4 SA-mutant Rad (l), in the absence or presence of exogenous PKA catalytic subunit. Dashed line is maximal P O for control of α 1C + β 2B without Rad. Scale bars: 1 pA and 25 ms. Control, n= 10; Control + PKA, n=5; Rad, n=5; Rad + PKA, n=8; 4SA mutant Rad, n=6, 4SA mutant Rad + PKA, n=5.

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Two Tailed Test, Mutagenesis, Control

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet:

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Transfection

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet:

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels

doi: 10.1172/JCI176943

Figure Lengend Snippet: ( A ) Schematic depicting Ca V β domains. NT, N-terminus; CT, C-terminus; Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a variable and flexible HOOK region that connects them. In β 2 -null mice, a frame-shift insertion causes an early termination at the end of the SH3 domain. ( B ) Anti-β 2 and anti-FLAG immunoblots. ( C ) Anti-FLAG and anti-β 2 immunofluorescence of nontransgenic WT and transgenic (TG) FLAG-β–expressing cardiomyocytes. Nuclear staining with DAPI. Scale bars: 50 μm. Representative of 3 similar experiments. Insets, ×4 enlargement to show striated pattern of expression. ( D – G ) Exemplar current-voltage relationships of Ca 2+ channels in the absence (black trace) and presence of forskolin (blue trace). Insets: Exemplar whole-cell Ca V 1.2 currents. Pulses from –60 mV to +10 mV before (black traces) and 3 minutes after (blue traces) forskolin. Horizontal scale bars = 50 ms, vertical scale bars = 10 pA/pF. ( H ) Fold change at –20 mV in peak current caused by forskolin (FSK). Mean ± SEM. P = not significant (NS) by 1-way ANOVA; n = 13, 16, 14, and 17 cardiomyocytes from 7, 3, 4, and 6 mice. TG, transgenic. ( I ) Boltzmann function parameter, V 50 , before and after FSK. Mean ± SEM. Statistical analysis among non-TG, β 2 , β 3 , and β 4 : P < 0.0001 by 1-way ANOVA; *** P < 0.001 by Šidák’s multiple-comparison test. Statistical analysis of no FSK versus FSK: **** P < 0.0001 by paired, 2-tailed t test. ( J ) Field stimulation–induced change in sarcomere contraction. **** P < 0.0001 by paired, 2-tailed t test. ( K ) Forskolin-induced fold change in sarcomere length. Mean ± SEM. n = 20, 20, 28, and 19 from 3, 3, 3, and 3 mice, from left to right. P = not significant by 1-way ANOVA.

Article Snippet: Proteins were size-separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with either a custom-made rabbit polyclonal anti–α 1C subunit antibody (Yenzym; 1:1,000 dilution) ( , ), a custom-made polyclonal anti–β subunit antibody , a custom-made polyclonal anti-Rad antibody (epitope: GSRGAGRERDRRRG, 1:1,000 dilution; Yenzym), anti–β-actin antibody (Santa Cruz Biotechnology, sc-47778; 1:1,000 dilution), and anti-FLAG antibody (Sigma-Aldrich, F7425; 1:1,000 dilution).

Techniques: Western Blot, Immunofluorescence, Transgenic Assay, Expressing, Staining, Comparison

Journal: JCI Insight

Article Title: Increased Ca 2+ influx through Ca V 1.2 drives aortic valve calcification

doi: 10.1172/jci.insight.155569

Figure Lengend Snippet: ( A and B ) IHC analysis of Ca V 1.2 (red) and RUNX2 (green) of valve tissue excised from a patient with CAVD ( A ) or a valve excised from a heart harvested for transplant and without CAVD ( B ). The blue signal is DAPI, and merged images are shown in the right-most panels for each. ( C and D ) Merged images for 2 additional valves from patients with CAVD ( C ) and from valves excised from hearts harvested for transplant and without CAVD ( D ). Scale bars: 100 μm.

Article Snippet: After blocking for 1 hour, the sections were incubated with a custom-made polyclonal anti-α 1C (Ca V 1.2) antibody (Yenzym, 1:50 dilution) ( – ) and a monoclonal anti-Runx2 antibody (Abnova, H00000860-M05).

Techniques:

Journal: JCI Insight

Article Title: Increased Ca 2+ influx through Ca V 1.2 drives aortic valve calcification

doi: 10.1172/jci.insight.155569

Figure Lengend Snippet: Immunofluorescence for α-SMA ( A ), Sox9 ( B ), Aggrecan ( C ), and Runx2 ( D ) with accompanying light transmission and merged images of valves from Cre – control and Scx-Ca V 1.2 TS mice shows expression of the specific marker within the valve lesion but not in the unaffected valve. For each marker, examples from ≥3 samples. Scale bar: 100 μm.

Article Snippet: After blocking for 1 hour, the sections were incubated with a custom-made polyclonal anti-α 1C (Ca V 1.2) antibody (Yenzym, 1:50 dilution) ( – ) and a monoclonal anti-Runx2 antibody (Abnova, H00000860-M05).

Techniques: Immunofluorescence, Transmission Assay, Control, Expressing, Marker

Journal: Nature

Article Title: Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

doi: 10.1038/s41586-020-1947-z

Figure Lengend Snippet: (a) Immunofluorescence of isolated α 1C -APEX2 and β 2B -APEX2 cardiomyocytes exposed to biotin-phenol and H 2 O 2 . Representative of 13 and 8 cardiomyocytes from 2 and 3 mice respectively. Scale bar = 5 μm. (b) Immunofluorescence of tissue sections of Langendorff-perfused α 1C -APEX2 heart. Scale bars: upper −100 μm; lower − 5 μm; lower inset− 5 μm. Representative of 10 sections from 2 mice. (c) Immunoblots of biotin-labeled proteins from α 1C -APEX2 and β 2B -APEX2 mice cardiomyocytes. In contrast to Ca V 1.2 subunits, RyR2, Jph2 and CaM, K V 1.5 channels were not detected in streptavidin-pulldown. Blots representative of 3 independent experiments. (d) Schematic of workflow for isolated cardiomyocytes. (e) Volcano plot of fold-change for relative protein quantification by TMT mass spectrometry of α 1C -APEX2 samples. Data shown are means for 5 pairs of biologically-independent samples. Non-adjusted unpaired two-tailed t-test. Rad (red dot) is reduced by 50% and PKA catalytic subunit (green dot) is increased by 50%. (f) Same as (e) except cardiomyocytes from β 2B -APEX2 mice. Data shown are means for 3 pairs of biologically independent samples. Rad is reduced by 30% and PKA catalytic subunit is increased by 68%. (g) Schematic of protein labeling and workflow for Langendorff-perfused α 1C -APEX2 mice hearts. bpm= beats per minute. (h) Same as (d) except proteins from α 1C -APEX2 whole heart samples. Data shown are means for 10 hearts, 5 without isoproterenol and 5 with isoproterenol. Rad is reduced by 36% (i) Schematic of workflow for isolated cardiomyocytes from non-transgenic (NTG) mice. (j) Same as (e) except proteins isolated from non-transgenic (NTG) mice cardiomyocytes without biotinylation or pull-down. * single peptide ESFDSQSLINNQSK. Data shown are means for 4 pairs of biologically-independent samples. Rad (red dot) in whole heart is unchanged by isoproterenol. For source gel data, see .

Article Snippet: Proteins were size-separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-V5 antibody (Thermofisher, R960-25; 1:5000 dilution), a custom-made polyclonal anti-α 1C antibody (Yenzym, 1:1000 dilution) , , a custom-made polyclonal anti-β antibody (epitope: mouse residues 120-138: DSYTSRPSDSDVSLEEDRE; Yenzym, 1:1000 dilution), an anti-JPH2 antibody (Pierce, PA5-20642, lot# NG1583142; 1:1000 dilution), an anti-calmodulin antibody (Millipore Sigma 05-173; 1:1000 dilution), a custom-made anti-RyR2 antibody (1:5000 dilution) , and an anti-K V 1.5 antibody (Alomone, APC-150, Lot# APC004AN0850; 1:1000 dilution).

Techniques: Immunofluorescence, Isolation, Western Blot, Labeling, Mass Spectrometry, Two Tailed Test, Transgenic Assay