Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Western blot detection of immunoprecipitated BNIP3 using an α-PKA substrate antibody specific to the phosphorylated RRXS/T sequence, located at the BNIP3 C-terminus and adjacent to the transmembrane (TM) domain. Results shown for 4 cell types: (left to right) HEK 293 cells expressing exogenous BNIP3 (dimer, 60 kD), and A549, MDA-MD-231, and AU565 cells expressing endogenous BNIP3 (monomer, 30kD). Lane 1 of each Western blot contains the whole cell lysate (WCL). (B) LC-MS/MS analysis of BNIP3 phosphorylation in HEK 293 cells with normal or elevated cAMP (8-Bromo-cAMP), showing peptide coverage (gray) and phosphorylation sites (red). The TM domain is underlined. (C) Table of BNIP3 phosphopeptides identified by LC-MS/MS, showing the percent probability, ion charge, actual and observed masses, and mass error (Da and ppm) for each peptide. Peptides shown are from analysis of BNIP3 purified from HEK 293 cells with elevated cAMP levels. (D) Schematic of the BNIP3 protein sequence, showing each C-terminal mutation representing phosphomimetic or nonphosphorylated BNIP3. (E) Expression of BNIP3 phosphomutants from stable doxycycline-inducible HEK 293 Tet On cells, treated with doxycycline (Dox) for 48 hr. (F) Subcellular localization of BNIP3 phosphomutants, showing Western blot of cytosolic and mitochondrial fractions. (G) Alkaline extraction of mitochondria-associated proteins, showing alkaline extract of the mitochondrial pellet and the alkaline-resistant mitochondrial pellet from cells expressing each BNIP3 phosphomutant. All blots are representative of at least 3 independent experiments.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Western Blot, Immunoprecipitation, Sequencing, Expressing, Liquid Chromatography with Mass Spectroscopy, Purification, Mutagenesis

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Representative examples of mitochondrial morphology, examined via transmission electron microscopy. Black arrows denote healthy, elongated mitochondria and white arrows denote rounded, swollen mitochondria. Scale bar represents 2 μm. (B) Quantification of electron microscopy, showing the average mitochondrial area per field. At least 15 fields were examined per cell type. (C) Percent elongated mitochondria per field, quantified from at least 15 microscope fields per cell type. (D) Mitochondrial mass of HEK 293 cells expressing each BNIP3 mutant, measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of Mitotracker Green FM. (E) Mitochondrial protein levels in HEK 293 cells expressing each BNIP3 mutant, monitored by detection of mitochondrially-encoded cytochrome c oxidase subunit II (MT-CO2). For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Transmission Assay, Electron Microscopy, Microscopy, Expressing, Mutagenesis, Fluorescence

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Representative examples of JC1 dual color fluorescence, examined by confocal microscopy. Red JC1 fluorescence is localized to polarized mitochondria whereas green fluorescence is independent of membrane polarization. Scale bar represents 10 μm. Red JC1 insets, denoted by white outlines, provide examples of the mitochondrial network in cells expressing each BNIP3 phosphomutant. Scale bar for inset is 5μm. Experimental controls included treatment of HEK 293 cells with Oligomycin A (Oligo A) or FCCP to hyperpolarize and depolarize mitochondria, respectively. (B) Mitochondrial membrane potential (ΔΨm), quantified as a ratio of red:green JC1 fluorescence by flow cytometry, where the same positive and negative controls were used to confirm efficacy of the assay (C) Levels of reactive oxygen species (ROS), measured by flow cytometric analysis of DHE fluorescence. (D) ROS levels, quantified by DCF fluorescence. (E) Mitochondrial ROS, measured as a ratio of MitoSox mean fluorescence intensity/Mitotracker mean fluorescence intensity for cells expressing each BNIP3 phosphomutant. All bar graphs represent the results observed in at least 3 independent experiments. Significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between pairs of complementary BNIP3 mutants are shown in brackets.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Fluorescence, Confocal Microscopy, Expressing, Flow Cytometry, Mutagenesis

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Representative images of GFP-LC3 puncta in HEK 293 cells expressing each BNIP3 mutant, examined via confocal microscopy. At least 50 cells were examined per cell type, in 3 independent experiments. Rapamycin (Rap) was used as a positive control. Scale bar represents 10 μm. (B) Quantification of the number of GFP-LC3 puncta per cell. (C) Western blot analysis of autophagic flux, where cells expressing each BNIP3 phosphomutant were treated without or with 50 nM Bafilomycin A1 (BAF). Blots are representative of 4 independent experiments. Two exposures of the LC3 Western blot are provided to show levels of LC3-II (short exposure) and LC3-I (long exposure). (D) Representative images of HEK 293 cells probed with Lysotracker Red, examined by confocal microscopy. (E) Quantification of the number of lysotracker puncta per cell, where a minimum of 30 cells were examined in 3 independent experiments. Scale bar represents 10 μm. (F) Quantification of mean lysotracker fluorescence intensity, measured by flow cytometry in 3 independent experiments. For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Expressing, Mutagenesis, Confocal Microscopy, Positive Control, Western Blot, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Levels of ROS, measured by the mean fluorescence intensity of DHE. HEK 293 control cells and cells expressing WT BNIP3 for 48 hr were treated with 8-Br-cAMP for 0, 2, or 4hr immediately prior to analyzing DHE fluorescence by flow cytometry. (B) Percent Annexin V positive cells undergoing the same 8-Br-cAMP treatment as described in (A). (C) Mitochondrial mass of HEK 293 cells cultured in normoxia or hypoxia for 48 hr and with or without 8-Br-cAMP treatment for the last 6 hr of normoxia/hypoxia. (D) Mitochondrial membrane potential of cells treated as described in (C). For each assay, a minimum of 30,000 events were collected per sample by flow cytometry. Data represents results from at least 3 separate experiments. Significant differences between untreated control cells (without BNIP3) and cells expressing WT BNIP3 are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between treatment conditions are shown in brackets. (E) Phosphorylation of endogenous BNIP3 at T188 following nutrient deprivation for 0, 30, or 120 min in three cell types: A549, MDA-MB-231, and AU565 cells, detected by probing immunoprecipitated BNIP3 with an α-PKA substrate antibody. (F) Phosphorylation level of endogenous BNIP3 at T188 following hypoxia for 0, 6, 24, or 48 hr in three cell types: A549, MDA-MB-231, and AU565 carcinoma cells, detected by probing immunoprecipitated BNIP3 with an α-PKA substrate antibody. To account for the increased expression of BNIP3 during extended hypoxia, the relative level of T188 BNIP3 phosphorylation is provided below each lane. The phosphorylation level, which represents the ratio of BNIP3 detected by α-PKA substrate antibody/total BNIP3, is expressed relative to the 0 hr time point of each cell type.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Fluorescence, Expressing, Flow Cytometry, Cell Culture, Immunoprecipitation

Journal: Translational Psychiatry

Article Title: Immunoglobulin G modulation of the melanocortin 4 receptor signaling in obesity and eating disorders

doi: 10.1038/s41398-019-0422-9

Figure Lengend Snippet: a Representative images of hMC4R GFP+ HEK 293 cells (green) 30 min after application of DyLight 550®-labeled (red) α-MSH affinity-purified IgG from eating disorder (anorexia nervosa, n = 9; bulimia nervosa, n = 7; binge eating disorder, n = 7), obese ( n = 10), and Ctrl ( n = 9) preincubated or not with α-MSH. Quantification of DyLight 550®-positive spots in hMC4R+ HEK 293 cells ( n = 50/group): b at the membrane; c intracellularly (cytosolic), and d ratios of cytosolic/membrane staining. Affinity kinetics properties of α-MSH/IgG IC for hMC4R + HEK 293 cells including e dissociation equilibrium constant (KD); f association rate (ka), and g dissociation rate (kd). Data are means ± s.e.m. Kruskal–Wallis test with Dunns’ post-tests ( b – d , f , g ) or analysis of variance with Tukey’s post-test ( e ), *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: MC4R-expressing HEK 293 cells (AMS Biotechnology, Abingdon, UK) were cultured in glass bottom Petri dishes (MatTek, ≈250,000 cells/dish).

Techniques: Labeling, Affinity Purification, Staining

Journal: Translational Psychiatry

Article Title: Immunoglobulin G modulation of the melanocortin 4 receptor signaling in obesity and eating disorders

doi: 10.1038/s41398-019-0422-9

Figure Lengend Snippet: a cAMP dose–response curves to α-MSH alone or α-MSH/IgG IC formed by IgG pooled in patents and control groups and adjusted to α-MSH-reactive IgG plasma levels of controls. cAMP dose–response curves to α-MSH preincubated or not with individual total IgG and corresponding EC50 ( b ) and maximal cAMP production ( c ). d Control experiments including cAMP dose–response curves to α-MSH by MC4R-expressing and non-expressing control HEK 293 cells and to α-MSH 1–4 peptide by MC4R-expressing cells ( n = 4). e cAMP dose-response curves to α-MSH and IgG from patients and controls without their overnight pre-incubation. f , cAMP dose–response curves to α-MSH alone or α-MSH/IgG IC co-administered (solid line) with agouti-related protein (AgRP; 100 nM) or added after AgRP preincubation (dotted line— n = 2/group) as well as in g cAMP maximal response. h , i cAMP dose–response curves of α-MSH preincubated with h purified total IgG from patients and controls depleted for α-MSH-reactive IgG ( n = 3/group) and i affinity-purified α-MSH-reactive IgG ( n = 6/group); j EC 50 and k maximal cAMP production at the plateau. Data are means ± s.e.m. Analysis of variance with Tukey’s post-test ( b , c , g , j ) or Kruskal–Wallis test with Dunns’ post-tests ( m ), *** p < 0.001, ** p < 0.01, * p < 0.05; Mann–Whitney test, $ p < 0.05. a α-MSH ( n = 9), Ctrl, anorexia nervosa (AN), bulimia nervosa (BN), and binge eating disorder (BED; n = 6), obese (OB; n = 4); d HEK 293-hMC4R+ ( n = 5), HEK 293-CTRL ( n = 6); e α-MSH ( n = 3), Ctrl, BN, and BED ( n = 2), AN and OB ( n = 3)

Article Snippet: MC4R-expressing HEK 293 cells (AMS Biotechnology, Abingdon, UK) were cultured in glass bottom Petri dishes (MatTek, ≈250,000 cells/dish).

Techniques: Expressing, Incubation, Purification, Affinity Purification, MANN-WHITNEY

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. HEK-293 cells overexpressing distinct α-syn constructs were lysed 48 h after transfection, and the cell lysates were analysed by immunoblotting for α-syn (Syn-1), without boiling. GAPDH was used as the loading control. A representative blot of n=3 experiments is shown B. Densitometry of data shown in (A.); a significant difference was indicated (p=0.045) by one-way ANOVA followed by a Tukey test for BiFC (Vn-α-syn) vs. BiFC (α-syn-Vc). C. WB of lysates from HEK-293 cells transfected with various amounts of vector α-syn DNA (depicted above the blot). α-syn was detected by anti-α-syn (Syn-1). The samples were boiled to disrupt complex formation to allow accurate determination of the levels of the two fragments. D. Relative protein levels (α-syn/β-tubulin) of BiFC proteins determined by quantification of the respective bands in (D) using Image Studio Lite. Data are shown as the mean of three independent experiments, each performed as technical duplicates (error bars represent the S.D.). E. Schematic of bidirectional inducible plasmid for expression of Vn-α-syn & α-syn-Vc under the control of a single tetracycline promoter. Immunoblot showing expression of both Vn-α-syn & α-syn-Vc upon addition of 1 μg /mL doxycycline. Samples were boiled prior to SDS-PAGE. No relevant significant difference was observed between the tested conditions using one-way ANOVA followed by a Tukey test. Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length)

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Construct, Transfection, Western Blot, Plasmid Preparation, Expressing, SDS Page

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. Size exclusion chromatography of a HEK-293 cell lysate 48 h post-transfection with the indicated α-syn constructs. The SEC fractions were analyzed by WB using anti-α-syn (Syn-1) antibodies. B. Calculated apparent MW depicted with a log scale plotted against the Kav value of each α-syn construct (large colored dots). Mixed circles represent either Vn-α-syn (red) or α-syn-Vc (blue) when being co-expressed. C. Standard curve generated using the indicated six reference proteins. D. Working model suggesting that the high propensity of Vn-α-syn to oligomerize and how such oligomers could interact with or entrap α-syn-Vc. Note, the UV280 signal in A does not correspond to the α-syn signal, but mostly proteins in the remaining cell lysate. Representative data shown from n=3 experiments. Vo: dead volume; Ve: elution volume; Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length).

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Size-exclusion Chromatography, Transfection, Construct, Generated

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. Living HEK-293 cells were crosslinked by the addition of DSG 48 h post transfection with the indicated α-syn constructs, and the resulting cell lysate was analysed by Western blotting against α-syn using the Syn-1 antibody. All analysed α-syn proteins displayed higher MWs upon addition of the DSG crosslinker, except for α-syn-Vc. B. The physiological dimer DJ-1 was used as an internal control so that the correct crosslinker concentration was applied. DJ-1 immunoblotting confirms that dimers are only detected upon crosslinking. C. Immunoblot of DSG crosslinked to live SH-SY5Y cells following 24 h BacMam delivery of either Vn-α-syn or α-Syn-Vc or both constructs. All displayed samples treated with DSG. Cells treated with only Vn-α-syn displayed higher order crosslinked oligomeric species in addition to monomeric Vn-α-syn. Cells treated with only α-syn-Vc displayed only monomeric α-syn-Vc. Treatment with both Vn-α-syn and α-Syn-Vc displayed higher order crosslinked oligomeric species in addition to both monomeric proteins. To determine if the higher order crosslinked oligomeric species in the sample treated with both Vn-α-syn and α-Syn-Vc were comprised of both proteins a separate WB for the FLAG epitope present only in the α-Syn-Vc protein was performed, this revealed the higher order crosslinked oligomeric species were predominantly comprised of Vn-α-syn. EV: empty vector; Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length). Blots are representative of n=3 experiments.

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Transfection, Construct, Western Blot, Concentration Assay, FLAG-tag, Plasmid Preparation

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. HEK-293 cells overexpressing distinct α-syn constructs were lysed 48 h after transfection, and the cell lysates were separated into a soluble and insoluble fraction by centrifugation. The resulting fractions were analysed by immunoblotting of α-syn (Syn-1). GAPDH was used as the loading control. Shown is a representative blot of n=3 experiments. B. Resulting soluble and insoluble band intensities of were quantified and normalized to soluble GAPDH. Significant differences were indicated (p<0.005) by one-way ANOVA followed by a Tukey test. C. Ratio of insoluble to soluble α-syn of Vn-α-syn and α-syn. Vc EV: empty vector; Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length).

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Construct, Transfection, Centrifugation, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: HEK-293 cells expressing the indicated α-syn constructs were fixed 48 h post-transfection and stained with the Syn-1 antibody. Upon co-transfection of the Vn-α-syn and α-syn-Vc constructs, Venus was successfully reconstituted and emitted a fluorescent signal that colocalized with the signal for α-syn. Mouse primary neurons were treated by PFF α-syn for 14 days and are positive for pS129. The nucleus was counterstained with DAPI. The scale bar is 20 μm in all the non-zoomed images and 10 μm for the zoomed images. The microscopy settings were kept the same among each construct. Shown are representative images from three independent experiments.

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Expressing, Construct, Transfection, Staining, Cotransfection, Microscopy

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: HEK-293 cells expressing the indicated α-syn constructs were fixed 48 h post-transfection and stained with the Syn-1 antibody. Upon co-transfection of the Vn-α-syn and α-syn-Vc constructs, Venus was successfully reconstituted and emitted a fluorescent signal that colocalized with the signal for α-syn. Mouse primary neurons were treated by recombinant pre-formed-fibrils α-syn for 14 days and are positive for the amytracker. The nucleus was counterstained with DAPI. MAP2 was used as a neuronal marker for the primary neurons and also colocalize with the amytracker. Both α-syn-V and the reconstituted BiFC fragments showed a nuclear localization and signal that did not colocalize with that of α-syn (Syn-1) The scale bar is 20 μm in all the non-zoomed images and 10 μm for the zoomed ones. Shown are representative images from two independent experiments. The microscopy settings were kept the same among each construct.

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Expressing, Construct, Transfection, Staining, Cotransfection, Recombinant, Marker, Microscopy

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of the preparatory and analytical workflows. (A) POMC derived ligands and their downstream signaling cascades. (B) Schematic overview of the thermal proteome profiling (TPP) workflow. MC3R-expressing HEK293 cells were treated with ACTH, α-MSH, or γ-MSH at concentrations of 20, 100, and 500 nM or with DMSO as a vehicle-only negative control. (C) Schematic overview of the TPP data analysis workflow. Protein identification and relative quantification were achieved by direct analysis of the raw LC–MS data, after which various bioinformatics tools were used to infer changes in transcription factor (TF) activity, perform enriched pathway analysis, and identify thermally affected proteins.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Derivative Assay, Expressing, Negative Control, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of identified proteins and thermally stabilized or destabilized proteins. (A) Venn diagrams showing the numbers of proteins exhibiting altered melting points, associations with enriched pathways, and phosphorylation in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH. (B) Venn diagrams showing the numbers of stabilized, destabilized, and phosphorylated proteins after incubation with ACTH, α-MSH, and γ-MSH. (C) Upset plot representing individual numbers of stabilized and destabilized proteins for each ligand and those common between various combinations of ligands.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Phospho-proteomics, Expressing, Incubation

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Characterization of transcription factors. (A) Heat map showing the relative abundance (compared to vehicle-only controls) of the transcription factors CCAR2, HMGB2, DDX21, SRSF7, and TET2 in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH at different ligand concentrations and temperatures. (B) Phosphorylation of tryptic peptides derived from the thermally stabilized and destabilized transcription factors shown in panel A whose activity was inferred to change following stimulation with ACTH, α-MSH, or γ-MSH. Phosphorylation sites are indicated by asterisks next to the modified amino acid (shown in parentheses when the exact amino acid is unknown). (C) Transcription factor activities and relational networks inferred from differential expression data using BITFAM. The heatmap shows fold changes in transcription factor activities (relative to vehicle-only treatments) in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, or γ-MSH. (D) Network showing the interconnectivity of the transcription factors identified within our experimental LC–MS data set.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Expressing, Incubation, Phospho-proteomics, Derivative Assay, Activity Assay, Modification, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk

doi: 10.1101/2023.08.28.555210

Figure Lengend Snippet: (A-C) Representative traces showing intracellular Ca 2+ responses to TrkB activation by BDNF (A), mGluR5 activation by glutamate (B) and TrkB activation by BDNF in mGluR5 co-expressing cells (C). (D) Distribution of BDNF responses in the absence or presence of mGluR5 co-expression. Only cells showing a response to glutamate are analyzed in the TrkB + mGluR5 condition. (E-G) Representative traces showing Ca 2+ responses to extended 30 min BDNF application in cells expressing TrkB (E) or TrkB and mGluR5 (F), with summary histogram (G) showing the distribution of response durations. (H-I) Co-application of the mGluR5 PAM VU-29 enhances the response to low dose BDNF, as seen in a representative cell (H) and a summary bar graph of the percentage of cells responding to BDNF (I). Only cells responding to glutamate were including in the bar graph in (I). (J-K) Representative western blot (J) and quantification (K) of BDNF-induced ERK activation (p-ERK/ERK ratio at 15 min) in HEK 293-TrkB cells co-expressing mGluR5, showing an enhanced response in the presence of 500 nM VU-29. For (D) and (I), points represent values from individual movies taken from distinct coverslips. For (K), individual points represent value from individual blots. For all conditions, data comes from at least 3 separate cell preparations. Unpaired t-test for (I); One-way ANOVA with Tukey’s multiple comparisons for (K). All data shown as mean ± SEM; ** P < 0.01, *** P < 0.001. See also Figure S3 and S4.

Article Snippet: HEK 293 cells were purchased from ATCC (CRL-1573) and authenticated by Bio-Synthesis, Inc. HEK 293-TrkB stable cell line is as previously described .

Techniques: Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk

doi: 10.1101/2023.08.28.555210

Figure Lengend Snippet: (A) Summary bar graph showing that co-expression of Gα q enhances Ca 2+ responses upon low dose BDNF-induced TrkB activation in HEK 293-TrkB cells. (B) Representative trace showing Ca 2+ response upon co-application of BDNF and subthreshold dose of 5-HT in HEK 293 cells co-expressing TrkB-ΔPLC and 5-HT 2A R. (C) Summary bar graph showing percentage of cells responding to BDNF in HEK 293 cells co-expressing TrkBΔPLC and mGluR1 or 5-HT 2A R or mGluR2 with or without co-application of 5-HT or glutamate. (D-E) Representative traces showing intracellular Ca 2+ responses to endogenous EGFR activation by EGF in the absence (D) or presence (E) of mGluR5. (F) Distribution of EGF responses in the absence or presence of mGluR5 co-expression. (G-H) Representative traces showing intracellular Ca 2+ responses to TrkA activation by NGF in the absence (G) or presence (H) of mGluR5. (I) Distribution of NGF responses in the absence or presence of mGluR5 co-expression. (J-K) Representative traces showing intracellular Ca 2+ responses to IGF1R activation by IGF in the absence (J) or presence (K) of mGluR5. (L) Distribution of IGF responses in the absence or presence of mGluR5 co-expression. For (A) (C), (F), (I) and (L), points represent values from individual movies taken from distinct coverslips. For all conditions, data comes from at least 3 separate cell preparations. Only cells showing a response to glutamate are analyzed in the conditions with mGluR5. One-way ANOVA with Tukey’s multiple comparisons is used for (A) and (C). All data shown as mean ± SEM; *** P < 0.01, *** P < 0.001. See also Figure S8.

Article Snippet: HEK 293 cells were purchased from ATCC (CRL-1573) and authenticated by Bio-Synthesis, Inc. HEK 293-TrkB stable cell line is as previously described .

Techniques: Expressing, Activation Assay