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: 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: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Impact of PTCH1b 5'UTR size and CGG-repeat number in gene reporter assays. pGL3-Promoter-derived constructs containing PTCH1b 5'UTRs (188 or 300 nucleotides) fused to the Firefly luciferase (Fluc) were transiently transfected in HCT116 p53+/+ (A), MCF7 (B) and HEK 293T cells (C). Presented are the average fold inductions relative to the empty vector and standard deviations of at least 3 biological replicates. The luciferase values are expressed relative to the value obtained with an empty control vector. The same constructs were used to measure relative Fluc mRNA transcript levels in transiently transfected HCT116 p53+/+ (D), MCF7 (E) and HEK 293T cells (F). Presented is the average Fluc mRNA expression normalized for the average plasmid copy number and for relative cDNA synthesis efficiency as revealed by the amplification of GAPDH and B2M mRNAs. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Derivative Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Control, Expressing, cDNA Synthesis, Amplification

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Impact of PTCH1b 5'UTR size and CGG-repeat number on cap-independent translation. Bicistronic pRuF-derived constructs were used to study the potential of the PTCH1b 5'UTR to drive cap-independent translation of the Firefly luciferase gene in transiently transfected MCF7 (A) or HEK 293T cells (B). Presented are the average ratios and standard deviations between Firefly (Fluc) and Renilla luciferase (Rluc) relative light units, normalized with the results obtained for empty pRuF vector, and standard deviation of at least 3 replicates. The PTCH1b 5'UTR size and CGG-repeat number are indicated. (C, D) The bicistronic nature of the transcript expressed by pRuF constructs was estimated measuring the Fluc/Rluc mRNA ratio by a qPCR approach. A ratio equal to 1.0 would strongly argue against the presence of a cryptic promoter within cloned PTCH1b 5'UTR resulting in a monocistronic Fluc mRNA transcript. A pRuF-type plasmid with cloned c-MYC 5'UTR was used as a positive control. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Derivative Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Standard Deviation, Clone Assay, Positive Control

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: An internal ribosome entry site (IRES) motif maps in the 3' end of the PTCH1b 5'UTR. (A) Putative PTCH1b IRES motif cloned into pRuF-type plasmid is not sufficient to obtain Firefly luciferase (Fluc) activity observed with the complete PTCH1b 5'UTR in MCF7 cells, while the remaining part of PTCH1b 5'UTRs (188ΔIRES and 300ΔIRES) retains certain levels of IRES activity. (B) Similar results were obtained in HEK 293T cells. (C) The ratios between Fluc and Renilla luciferase (Rluc) mRNA for remodeled pRuF PTCH1b 5'UTR plasmids in transfected MCF7 cells didn't deviate from 1.0. (D) The same results were obtained in HEK 293T cells. Results are presented as described in Figure 4.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Hypoxia enhances PTCH1b 5'UTR mediated translation. MCF7 and HEK 293T cells transiently transfected with the different pRuF reporter constructs were grown in normoxic (dark gray) or severe hypoxic (light gray) conditions for 16 hours. Luciferase assays were performed as described in Materials and Methods and shown as those presented in Figure 5.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Transfection, Construct, Luciferase

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Higher PTCH1b mRNA relative translation efficiency during hypoxia in HEK 293T cells overexpressing GLI1. HEK 293T cells were transfected with an empty vector or a construct overexpressing GLI1 and then cultured in normoxia or hypoxia for 16 hours (48 hours post transfection). (A). Total RNA levels of the indicated mRNAs, quantified by qPCR as described in the method section and presented as relative expression compared to the reference genes (ΔCq). c-MYC mRNA was included as an example of mRNA whose 5'UTR is considered to possess IRES activity, while PCNA was included as negative control for IRES function. Error bars plot the average and standard deviations of 3 replicates (B). Cytoplasmic lysates of HEK 293T cells transfected and cultured as for panel A, were separated by sucrose-gradient equilibrium density; subpolysomal (SUB) and polysome-associated (POL) mRNAs were identified and collected by UVC scanning and the indicated transcripts were quantified by qPCR, as for panel A. (C). The results from panel B, are also plotted as ratio between polysome-associated and subpolysomal mRNAs as a function of the different treatment. This ratio is considered an estimate of relative mRNA translation efficiency. (D). Global relative mRNA translation rates in HEK 293T cells cultures in normoxia or hypoxia for 16 hours were assessed by incorporation of an immune-detectable methionine analog, as described in Materials and Methods. (E). Western blot analysis of PTC1-L protein and PCNA. Transfection of the GLI1 overexpression plasmid and treatment are indicated. Beta-tubulin was used as reference protein.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Transfection, Plasmid Preparation, Construct, Cell Culture, Expressing, Activity Assay, Negative Control, Western Blot, Over Expression

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Selective Cannabinoid 2 Receptor Stimulation Reduces Tubular Epithelial Cell Damage after Renal Ischemia-Reperfusion Injury

doi: 10.1124/jpet.117.245522

Figure Lengend Snippet: (A) Functional activities of SMM-295 in the CB2 ACTOne assay (open circles), CB1 ACTOne assay (open triangles), and parental ACTOne cells containing only the CNG ion channel (filled squares). The functional activation of CB2 by the internal control (CP 55,940; filled diamonds) is shown. (B) Western blot analysis of signaling proteins after brief exposure (15 minutes) to SMM-295 in rat NRK-52E proximal tubule epithelial cells. Activation of prosurvival Akt/PKB and MAPK (ERK1/2 and p38) was detected using specific antibodies. β-actin was used as a loading control. ERK1/2, extracellular signal–regulated kinase 1/2.

Article Snippet: Human embryonic kidney (HEK)–cyclic nucleotide gated (CNG), HEK-CNG+CB1, and HEK-CNG+CB2 cells were obtained from Codex BioSolutions.

Techniques: Functional Assay, Activation Assay, Western Blot