Journal: The Journal of General Physiology

Article Title: Proteomic and functional mapping of cardiac Na V 1.5 channel phosphorylation sites

doi: 10.1085/jgp.202012646

Figure Lengend Snippet: Phosphorylation sites, phosphopeptides, and site-discriminating ions identified in immunoprecipitated Na V 1.5 proteins from sham and TAC mouse left ventricles using MS

Article Snippet: Magnetic beads were then collected and washed rapidly four times with ice-cold lysis buffer, and isolated protein complexes were eluted from the beads in 1× SDS sample buffer (Bio-Rad Laboratories) at 60°C for 10 min. 99% of the immunoprecipitated mouse left ventricular Na V channel protein complexes were analyzed by MS, and the remaining 1% were used to verify IP yields by Western blotting using a rabbit polyclonal anti-Na V 1.5 antibody (RbαNa V 1.5, 1:1,000, ASC-005; Alomone Laboratories).

Techniques: Immunoprecipitation, Sequencing

Journal: The Journal of General Physiology

Article Title: Proteomic and functional mapping of cardiac Na V 1.5 channel phosphorylation sites

doi: 10.1085/jgp.202012646

Figure Lengend Snippet: Localization and quantification of 42 MS-identified Na V 1.5 phosphorylation sites in mαNa V PAN-IPs from sham and TAC mouse left ventricles (LVs). (A) Schematic representation of phosphorylation sites on the Na V 1.5 protein (UniProt reference sequence K3W4N7 ). Two phosphorylation site locations are possible at amino acids S1056-T1058. (B) The areas of extracted MS1 ion chromatograms, corresponding to MS2 spectra assigning phosphorylated (in red) and nonphosphorylated (in white) Na V 1.5 peptides at indicated phosphorylation site(s), in mαNa V PAN-IPs from sham and TAC LVs are indicated. No red color is visible for the phosphorylated peptide at position T1809, because this phosphopeptide area is very small (area = 80,291 arbitrary unit) relative to the areas of the nonphosphorylated peptides (areas = 80,060,220 arbitrary unit). (C) The areas of extracted MS1 ion chromatograms, corresponding to MS2 spectra assigning phosphorylated peptides at indicated phosphorylation site(s), in mαNa V PAN-IPs from sham and TAC LVs are indicated. The brackets indicate the subgroups of phosphorylation sites analyzed in B. Independent quantification of S459 and S460 phosphorylated peptides was not possible, because localization of the phosphorylation site in most of the phosphorylated peptides could not be discriminated. Similar to B, no red bar is visible for the phosphorylated peptide at position T1809, because this phosphopeptide area is very small (area = 80,291 arbitrary unit), relative to the areas of the other phosphorylated peptides. (D) Distributions and mean ± SEM relative abundances of individual Na V 1.5 phosphopeptides allowing assignments of indicated phosphorylation site(s), as well as of corresponding nonphosphorylated (NP) peptides, in TAC LV ( n = 5, in black) versus sham LV ( n = 4, in white) mαNa V PAN-IPs were obtained using TMT reporter ion intensities. The relative abundances of Na V 1.5 phosphopeptides exhibiting phosphorylation(s) on serine 671 (S671; n = 12 peptides) alone or in combination with serine 664 (S664 + S671; n = 9 peptides) or serine 667 (S667 + S671; n = 7 peptides) are increased (**, P < 0.01; ***, P < 0.001; Mann-Whitney test) in TAC LV versus sham LV mαNa V PAN-IPs. (E) Experimental workflow used in the study. Once immunoprecipitated using the mαNa V PAN antibodies, the Na V channel complexes from sham and TAC mouse LVs were labeled individually with different TMT 10 tags and combined in the same TMT set for multiplexed LC-MS/MS analysis. Na V 1.5 phosphorylation sites were identified, quantified, and analyzed by clusters in whole-cell voltage-clamp recordings in HEK-293 cells.

Article Snippet: Magnetic beads were then collected and washed rapidly four times with ice-cold lysis buffer, and isolated protein complexes were eluted from the beads in 1× SDS sample buffer (Bio-Rad Laboratories) at 60°C for 10 min. 99% of the immunoprecipitated mouse left ventricular Na V channel protein complexes were analyzed by MS, and the remaining 1% were used to verify IP yields by Western blotting using a rabbit polyclonal anti-Na V 1.5 antibody (RbαNa V 1.5, 1:1,000, ASC-005; Alomone Laboratories).

Techniques: Sequencing, MANN-WHITNEY, Immunoprecipitation, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of General Physiology

Article Title: Proteomic and functional mapping of cardiac Na V 1.5 channel phosphorylation sites

doi: 10.1085/jgp.202012646

Figure Lengend Snippet: Proteins identified in immunoprecipitated Na V channel complexes from sham and TAC mouse left ventricles using MS

Article Snippet: Magnetic beads were then collected and washed rapidly four times with ice-cold lysis buffer, and isolated protein complexes were eluted from the beads in 1× SDS sample buffer (Bio-Rad Laboratories) at 60°C for 10 min. 99% of the immunoprecipitated mouse left ventricular Na V channel protein complexes were analyzed by MS, and the remaining 1% were used to verify IP yields by Western blotting using a rabbit polyclonal anti-Na V 1.5 antibody (RbαNa V 1.5, 1:1,000, ASC-005; Alomone Laboratories).

Techniques: Immunoprecipitation, Sequencing

Journal: Autophagy

Article Title: Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

doi: 10.1080/15548627.2015.1096485

Figure Lengend Snippet: Defective autophagy in VSMCs elicits cellular hypertrophy, and increases migration capacity and total collagen amount. ( A ) VSMCs isolated from Atg7 +/+ Tagln-Cre + (+/+) and Atg7 F/F Tagln-Cre + (−/−) aorta were labeled with calcein AM and visualized by confocal fluorescence microscopy. Scale bar: 10 µm. Cell size was measured using z-stack images (*, P < 0.05; n = 2 experiments; Student t test). ( B ) Thoracic aorta of Atg7 +/+ and atg7 −/− mice were stained with H&E to measure the width of the media (white arrows) (**, P < 0.01; n = 6 regions/aorta; Univariate). Scale bar: 25 µm. Note that the number of VSMC layers between Atg7 +/+ and atg7 −/− aorta was not different. ( C ) Migratory capacity of Atg7 +/+ and atg7 −/− VSMCs was analyzed using an Oris Migration Assay (***, P < 0.001; n = 2 experiments in triplicate; Student t test). Western blot analysis of TGFB and CXCL12 in Atg7 +/+ and atg7 −/− VSMCs. ( D ) Atg7 +/+ and atg7 −/− VSMCs were left untreated or treated with 10 ng/ml TGFB for 48 h and stained with Sirius red to examine total collagen amount (***, P < 0.001 vs. Atg7 +/+ ; ### , P < 0.001 vs. control; n = 4 experiments in triplicate; two-way ANOVA with genotype and treatment as category factors).

Article Snippet: Membranes were probed with the following primary antibodies: goat anti-GSTA (ab53940), rabbit anti-NFE2L2 (ab137550), mouse anti-CDKN2A/p16 (ab54210) and rabbit anti-CDKN1A/p21 (ab7960) from Abcam; rabbit anti-CXCL12 (Bioss, bs-4938); rabbit anti-GAPDH (14C10), rabbit anti-TGFB (3711) and rabbit anti-phospho RB (8516) from Cell Signaling Technology; mouse anti-MAP1LC3B (Nanotools, clone 5F10, 0231-100); rabbit anti-NQO1 (Novus Biologicals, NBP1-40663); rabbit anti-PARP1 (sc-7150), rabbit anti-CDKN2A/p16 (sc-1207) and rabbit anti-total RB (sc-50) from Santa Cruz Biotechnology; mouse anti-ACTB (clone AC-15, A5441), rabbit anti-ATG7 (A2856), rabbit anti-ATG5 (A0856), rabbit anti-SQSTM1/p62 (P0067) and rabbit anti-acetyl-TP53 (SAB4503014) from Sigma-Aldrich.

Techniques: Migration, Isolation, Labeling, Fluorescence, Microscopy, Staining, Western Blot

Journal: Autophagy

Article Title: Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

doi: 10.1080/15548627.2015.1096485

Figure Lengend Snippet: Defective VSMC autophagy promotes upregulation of MMP9, TGFB and CXCL12, 5 d after ligation-induced injury. ( A ) The left common carotid artery (LCCA) of Atg7 +/+ Tagln-Cre + (+/+) and Atg7 F/F Tagln-Cre + (−/−) mice (n = 3) was ligated for 5 d. Gelatin zymographic analysis of the LCCA to detect MMP9 and MMP2 activity followed by densitometric analysis (***, P < 0.001; NS, not significant; Student t test). ( B ) Western blot analysis of the LCCA for TGFB, CXCL12 and GAPDH. Relative expression of TGFB/GAPDH and CXCL12/GAPDH was determined by densitometric analysis (*, P < 0.05; ***, P < 0.001; Student t test).

Article Snippet: Membranes were probed with the following primary antibodies: goat anti-GSTA (ab53940), rabbit anti-NFE2L2 (ab137550), mouse anti-CDKN2A/p16 (ab54210) and rabbit anti-CDKN1A/p21 (ab7960) from Abcam; rabbit anti-CXCL12 (Bioss, bs-4938); rabbit anti-GAPDH (14C10), rabbit anti-TGFB (3711) and rabbit anti-phospho RB (8516) from Cell Signaling Technology; mouse anti-MAP1LC3B (Nanotools, clone 5F10, 0231-100); rabbit anti-NQO1 (Novus Biologicals, NBP1-40663); rabbit anti-PARP1 (sc-7150), rabbit anti-CDKN2A/p16 (sc-1207) and rabbit anti-total RB (sc-50) from Santa Cruz Biotechnology; mouse anti-ACTB (clone AC-15, A5441), rabbit anti-ATG7 (A2856), rabbit anti-ATG5 (A0856), rabbit anti-SQSTM1/p62 (P0067) and rabbit anti-acetyl-TP53 (SAB4503014) from Sigma-Aldrich.

Techniques: Ligation, Activity Assay, Western Blot, Expressing

Journal: Autophagy

Article Title: Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

doi: 10.1080/15548627.2015.1096485

Figure Lengend Snippet: Overview of the mechanisms by which defective VSMC autophagy accelerates senescence and promotes postinjury neointima formation and diet-induced atherogenesis. SQSTM1 accumulation in autophagy defective VSMCs triggers NFE2L2 activation and transcription of multiple antioxidative enzymes including GSTA and NQO1. Upregulation of GSTA and NQO1 promotes VSMC survival against oxidative stress under defective autophagy conditions. SQSTM1 accumulation in autophagy defective VSMCs triggers the development of stress-induced premature senescence. Autophagy defective VSMCs are characterized by CDKN2A-RB-mediated G 1 proliferation arrest, increased migration and changes in VSMC phenotype. Enhanced migration is associated with increased secretion of MMP9, TGFB and CXCL12. The phenotype of autophagy defective VSMCs is defined by nuclear and cellular hypertrophy, and by increased collagen content. Defective autophagy in VSMCs accelerates postinjury neointima formation and diet-induced atherogenesis.

Article Snippet: Membranes were probed with the following primary antibodies: goat anti-GSTA (ab53940), rabbit anti-NFE2L2 (ab137550), mouse anti-CDKN2A/p16 (ab54210) and rabbit anti-CDKN1A/p21 (ab7960) from Abcam; rabbit anti-CXCL12 (Bioss, bs-4938); rabbit anti-GAPDH (14C10), rabbit anti-TGFB (3711) and rabbit anti-phospho RB (8516) from Cell Signaling Technology; mouse anti-MAP1LC3B (Nanotools, clone 5F10, 0231-100); rabbit anti-NQO1 (Novus Biologicals, NBP1-40663); rabbit anti-PARP1 (sc-7150), rabbit anti-CDKN2A/p16 (sc-1207) and rabbit anti-total RB (sc-50) from Santa Cruz Biotechnology; mouse anti-ACTB (clone AC-15, A5441), rabbit anti-ATG7 (A2856), rabbit anti-ATG5 (A0856), rabbit anti-SQSTM1/p62 (P0067) and rabbit anti-acetyl-TP53 (SAB4503014) from Sigma-Aldrich.

Techniques: Activation Assay, Migration

Journal: Journal of Biological Chemistry

Article Title: Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

doi: 10.1074/jbc.m805000200

Figure Lengend Snippet: FIGURE 7. Bioactivity of platelet-derived chemerin. A, comparison of che- motactic activity of chemerin released from activated platelets, prochemerin, and the active form chemerin21–157. B, the specific chemotactic response of platelet-derived chemerin to CMKLR1/L1.2 transfectants. C, proteolytic regu- lation of platelet-derived chemerin bioactivity. Platelet-derived chemerin was partially purified by heparin affinity chromatography (8). Fractions con- taining chemerin were eluted with 0.6 M of NaCl. The concentration of chemerin was quantitated by ELISA. The conditions of platelet-derived chemerin treated with CPN, CPB, plasmin, plasmin/CPN, or plasmin/CPB were identical to that in Fig. 3. The results represent one of three independent experiments and are expressed as the means S.D. (n 3). *, p 0.05; **, p 0.005.

Article Snippet: Materials—Recombinant human chemerin21–157, polyclonal goat anti-human chemerin antibodies, and biotinylated polyclonal goat anti-human antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Derivative Assay, Comparison, Activity Assay, Purification, Affinity Chromatography, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 4. Stimulation of IVD cells with HTRA1-generated fibronectin fragments. A, concentrated protein supernatants (15 g) from IVD cells treated for 24 h without or with HTRA1mac (5 g/ml) or HTRA1macSA (5 g/ml) were subjected to immunoblotting using antibody Mab1935 specific for the fibronectincarboxyl-terminalheparin-bindingdomain(Cterminus)orMab1936specificforthefibronectinamino-terminalfibrin-andheparin-bindingdomain (N terminus). Fibronectin fragments containing the amino-terminal fibrin- and heparin-binding domain are identified by the closed arrowhead. B, purified human plasma-derived fibronectin (Fn) was incubated with HTRA1mac or HTRA1macSA at equimolar concentrations in TBS, pH 8.5, for 16 h at 37 °C, and samples were loaded onto a 4–15% gradient gel and stained with Coomassie Blue. Fibronectin and recombinant HTRA1 alone were also loaded and served as controls. C, an equimolar concentration of human plasma-derived fibronectin and HTRA1mac were incubated for 16 h, and fibronectin fragments were visualized by Western blot analysis using the antibodies described in A. D, equimolar concentrations of fibronectin (20 g) and HTRA1mac (5 g) were incubated for 16 h, and fibronectin fragments were purified by affinity chromatography. IVD cells were incubated with purified HTRA1-digested fibronectin (FnHTRA1mac) for 24 h, and expression levels of MMP1, MMP3, and MMP13 mRNA were determined by qRT-PCR and the -fold change as compared with untreatedcontrolswasdeterminedusingthe2CTmethod.Additionalcultureswereincubatedwitheitheraffinity-purifiedTris-bufferedsaline,pH7.6(TBS), fibronectin (Fn), or HTRA1 (HTRA1mac) or left untreated (Control). Data are representative of two separate experiments performed using IVD cells from two patients. Shown are results of triplicate determinations S.D. *, p 0.01, as determined by one-way ANOVA.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Generated, Western Blot, Binding Assay, Purification, Clinical Proteomics, Derivative Assay, Incubation, Staining, Recombinant, Concentration Assay, Affinity Chromatography, Expressing, Quantitative RT-PCR, Control

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 5. Detection of fibronectin fragments in degenerated IVD tissue. A, fibronectin (FN) mRNA levels in intact IVD tissue samples from patients (n 36) with varying degrees of IVD degeneration were determined by qRT-PCR and presented as 2CT S.E. (error bars). B, correlation study between FN and HTRA1 mRNA levels (2CT) in patient IVD tissue samples (n 36). R2, square of correlation coefficient; p 0.01 as determined from Pearson’s correlation coefficient. C, protein extracts from patient IVD tissues (n 12) were loaded onto a 12% SDS-polyacrylamide gel, and immunoblotting was performed using a monoclonal antibody (Mab1936) specific for the amino-terminal fibrin- and heparin-binding domain. D, the PVDF membrane used in C was stained with Coomassie Blue in order to confirm equal protein loading. Lane 1, HTRA1-digested human plasma-derived fibronectin; lanes 2–4, non-degenerated (ND) discs; lanes 5–7, mildly degenerated discs; lanes 8–10, moderately degenerated discs; lanes 11–13, severely degenerated discs.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Quantitative RT-PCR, Western Blot, Binding Assay, Membrane, Staining, Clinical Proteomics, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 6. A theoretical model for the role of HTRA1 in IVD degeneration. Based on our findings, we propose that HTRA1 accumulates in IVD tissue undergoing degeneration and stimulates MMP production by resident cells in a predominantly protease-dependent manner, via activation of the MEK pathway. Furthermore, we suggest that the stimulatory effects of HTRA1 on IVD cells are mediated indirectly through its ability to generate fibronectin fragments, although other routes of cellular activation cannot be ruled out. IDD, intervertebral disc degeneration.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Activation Assay

Journal: Cell Metabolism

Article Title: Nutrient-sensing AgRP neurons relay control of liver autophagy during energy deprivation

doi: 10.1016/j.cmet.2023.03.019

Figure Lengend Snippet:

Article Snippet: Sections were then mounted with DAPI using Vectashield Antifade Mounting Medium (Vector Laboratories), covered using a coverslip and stored at 4°C in the dark.

Techniques: Virus, Plasmid Preparation, Recombinant, Protease Inhibitor, Western Blot, Blocking Assay, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Bicinchoninic Acid Protein Assay, Reverse Transcription, RNAscope, Multiplex Assay, Software, Microscopy, Mass Spectrometry, Liquid Chromatography, Chromatography