Journal: American journal of human genetics

Article Title: Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82.

doi: 10.1016/j.ajhg.2010.05.010

Figure Lengend Snippet: Figure 2. Localization of Gpsm2 in the Mouse Inner Ear (A and B) Schematic representation of the cochlea (A) and utricle (B), one of the vestibular organs. The inner and outer hair cells of the cochlea make up the sensory cells; they are embedded in non- sensory supporting cells, including the Dei- ters, pillar, and Hensen cells. The utricle alsocontainshaircellsandsupporting cells. (C–J) Immunofluorescence images of the cochlea at e16.5 (C and D) and p0 (G and H) and of the utricle at e16.5 (F) and p0 (J). Gpsm2 (green) is localized at the apical surface of the hair and supporting cells, both in the cochlea and utricle and along the greater epithelial ridge (E and I) in e16.5 and p0 inner ears. In p0 cochlea, it is specifically localized at the lateral ridge of the hair cell surface. It is also localized in the pillar cells at this age. Hair cell cyto- plasm is marked by myosin VI (red), and the nuclei are marked by DAPI (blue). Paraffin embedded sections were stained with primary antibodies goat anti-myosin VI (Santa Cruz Biotechnology) and rabbit anti-Gpsm2 (ProteinTech; for validation of the antibody, see 12) and the fluores- cence-conjugated secondary antibodies donkey anti-rabbit 488 (Molecular Probes) and donkey anti-goat cy3 (Sigma). Imaging was done with an LSM 510 confocal micro- scope (Zeiss). Scale bars represent 5 mm (C, E–G, I, and J), 2 mm (D), and 2.5 mm (H). All procedures involving animals met NIH guidelines and were approved by the Animal Care and Use Committees of Tel Aviv University and the University of Washington.

Article Snippet: We evaluated inner ear sections from embryonic day 16.5 (e16.5), postnatal day 0 (p0), p15, and p60 mice by immunohistochemistry 92 The American Journal of Human Genetics 87, 90–94, July 9, 2010 with an antibody against Gpsm212 (ProteinTech) (Figure 2; Figure S2).

Techniques: Staining, Biomarker Discovery, Imaging

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 2. YAP1 and TAZ regulate expression of SLC38A1 and SLC7A5 in HCC cells. (A) Expression pattern of SLC family genes from microarray data. Amino acid transporters are shown in red. (B-E) The indicated cells were transfected with the indicated siRNAs. Cells were used for quanti- tative RT-PCR to measure SLC38A1 and SLC7A5. Expression level was normalized with siLuc samples. (F) Quantitative RT-PCR was done with Mst1/2 knockout samples to measure connective tissue growth factor, SLC38A1, and SLC7A5 messenger RNA expression level. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: CTGF, connective tissue growth factor; Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Microarray, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Knock-Out, RNA Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 4. Amino acid transporters are important for proliferation of HCC cells. (A-D) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs, and cell proliferation rates were measured by an MTT assay at the indicated time points. Values shown were normalized to siLuc-treated cells and represent mean 6 standard deviation. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E) Recovered cell growth by exogenous SLC38A1 and SLC7A5 in YAP1/TAZ-depleted SK-Hep1 cells. Myc-tagged exogenous SLC38A1 and SLC7A5 were expressed in SK-Hep1 cells. Empty expression vector pCMV6 was used as a control. Cell proliferation rates were measured by the MTT assay at 72 hours after transfection of expression vectors. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (F,G) Tumor weight after treat- ment with siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in a subcutaneous xenograft model (F) or an orthotopic xenograft model (G) with SK-Hep1 HCC cells. At 6 weeks after siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine injection, mice were killed and tumor weights measured (n 5 10 per group). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: MTT Assay, Standard Deviation, Expressing, Plasmid Preparation, Control, Transfection, Injection

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 5. YAP1 and TAZ directly bind to the promoters of amino acid transporters. (A) Schematic representation of SLC38A1 promoter regions for ChIP assay (top) and ChIP assay results (bottom). (B) Schematic representation of SLC7A5 promoter regions for ChIP assay (top) and ChIP assay results (bottom). ChIP assay was done in SK-Hep1 cells with indicated antibodies. Recruitment of YAP1, TAZ, and TEAD1 proteins to the SLC38A1 or SLC7A5 promoter was analyzed using primers specific to the SLC38A1 or SLC7A5 promoter. Immunoglobulin G was used as an internal control. (C,D) ChIP samples were used for quantitative RT-PCR for quantification of binding in indicated cell lines. Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E,F) SK-Hep1 and SNU-449 cells were transi- ently transfected with indicated complementary DNA and reporter plasmids, and luciferase activity was measured by a luminometer (Promega). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: IgG, immunoglobulin G; IP, immunoprecipitation; TBS, TEAD binding site.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Control, Quantitative RT-PCR, Binding Assay, Transfection, Luciferase, Activity Assay, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 6. mTORC1 is regulated by SLC38A1 and SLC7A5 in HCC cells. (A) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs for 96 hours. Phosphorylation of S6K1 was assessed by western blotting with the indicated antibodies. (B) SK-Hep1 cells were treated with esterified glutamine (Gln, 4 mM) for 30 minutes after silencing of SLC38A1 with specific siRNA. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with indicated antibodies. (C,D) Xeno- grafted HCC tissues treated with the indicated siRNAs were used for western blot analysis with the indi- cated antibodies.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 7. YAP1 and TAZ activate mTORC1 through regulation of SLC38A1 and SLC7A5 (A,B). SK- Hep1 (A) and SNU-449 (B) HCC cells were transiently transfected with the indicated siRNA for 96 hours. Expression of YAP1 and TAZ and phosphorylation of S6K1 and S6 were assessed by western blot- ting with the indicated antibodies. (C) SK-Hep1 cells were transfected with SLC38A1 and SLC7A5 com- plementary DNA after silencing YAP1 and TAZ with specific siRNAs. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with the indicated antibodies. (D) Expres- sion of Mst1, Mst2, S6K1, and S6 and phosphorylation of S6K1 and S6 were assessed in liver tissues from wild-type and Mst1/2-/- mice (4 weeks old) by western blotting with indicated antibodies. Abbrevia- tion: Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Transfection, Expressing, Phospho-proteomics, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 8. Significance of YAP1/TAZ-mediated regulation of mTORC1 in human HCC and mouse HCC models. (A,B) Kaplan-Meier plots of overall survival of HCC patients in cohort 1. Patients were stratified according to expression level of SLC38A1 (A) or SLC7A5 (B). P values were calculated with the log-rank test. (C) Liver tumor numbers after rapamycin treatment in Mst1/2-/- mice. At 62 days after rapamycin treatment, mice were killed and tumors counted. Data are presented as mean 6 standard error of the mean. (D) Representative images of liver tissue treated with rapamycin or vehicle. (E) Expression and phosphorylation of mTOR1 downstream targets in HCC tissues assessed by western blotting with the indicated antibodies after rapamycin or vehicle treatment in Mst1/2-/-

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer.

doi: 10.1186/s13046-022-02330-4

Figure Lengend Snippet: Fig. 1 Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, CD98hc and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in (C). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in (F). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown

Article Snippet: Exploration of commercial sources of antibodies using the Antibodypedia online tool led to the identification of an anti-SLC7A5/ LAT1 antibody (BU53, Novus Biologicals) and an antiGLUT1 antibody (MAB1418, R&D Systems, MN, USA) that could meet these properties.

Techniques: Microarray, Clinical Proteomics, Membrane, Gene Expression, Expressing, Western Blot, Control, Quantitation Assay, Immunoprecipitation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer.

doi: 10.1186/s13046-022-02330-4

Figure Lengend Snippet: Fig. 4 Generation and anti-proliferative activity of an antibody-drug conjugate targeting CD98hc. A Preparation of the antibody-drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hc antibody was analyzed by Western, by using an anti-DM1 antibody. Twenty nanograms of this ADC (αCD98hc-DM1), the nude anti-CD98hc (αCD98hc), trastuzumab or T-DM1 were loaded in 12% SDS-PAGE gels and analyzed for total protein (stain-free blot, lower image) and DM1 reactivity (upper panel). Trastuzumab and T-DM1 were used as a negative and positive controls. B Effect of anti-CD98hc-DM1 in a panel of TNBC cell lines. Cells were treated with anti-CD98hc and anti-CD98hc-DM1 10 nM for four days. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of triplicates of an experiment repeated twice. C Dose-response analyses of the effect of anti-CD98hc-DM1 on four TNBC cell lines. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D and E HS578T (D) and MDA-MB231 (E) cells were treated with anti-CD98hc, anti-CD98hc-DM1 or DM1 for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. F Knockout of CD98hc in MDA-MB231 cells by CRISPR/Cas9. Parental MDA-MB231 cells and two different clones knocked out for CD98hc were lysed. The levels of expression of CD98hc and LAT1 were analyzed by Western blot. Calnexin was used as a loading control. G Dose-response analyses of the effect of anti-CD98hc-DM1 on parental and CD98hc CRISPR #B3, #G3 MDA-MB231 cells. Cells were treated with anti-CD98hc-DM1 for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times

Article Snippet: Exploration of commercial sources of antibodies using the Antibodypedia online tool led to the identification of an anti-SLC7A5/ LAT1 antibody (BU53, Novus Biologicals) and an antiGLUT1 antibody (MAB1418, R&D Systems, MN, USA) that could meet these properties.

Techniques: Activity Assay, Western Blot, SDS Page, Staining, Control, Knock-Out, CRISPR, Clone Assay, Expressing

Journal: Nature Communications

Article Title: Intrinsic resistance to RAS inhibitors is driven by dysregulation of KRAS degradation

doi: 10.1038/s41467-025-67109-5

Figure Lengend Snippet: A , B Heatmaps show the proteomic profiles of proteins in proximity to KRAS, which served as the bait in proximity labeling experiments , . C Immunoblot analysis of the indicated proteins in H727 cells expressing the indicated shRNAs. SLC3A2 (or SLC7A5), p70 S6K p-T389, and Vinculin were detected on the same gel, but different gels for total p70 S6K that was processed in parallel. The immunoblot quantification presented as mean ± SEM; n = 4 technical replicates per group. D VEGFA mRNA levels measured using qPCR in the supernatant of the indicated H727 cells. Data are shown as mean ± SEM, n = 4 technical replicates per group. E ELISA-based VEGFA levels in the supernatant of the indicated H727 cells. Data are shown as mean ± SEM, n = 4 technical replicates per group. F Immunoblotting analysis of SLC7A5 and SCL3A2 expression in DMSO- or RMC7977 (10 nM, 24 hours)-treated H727 cells expressing sh GFP or sh LZTR1 . SLC3A2, SLC7A5, and Vinculin were detected on the same gel. G Immunoblotting analysis of MTOR, SLC7A5 and SCL3A2 expression after lysosome enrichment using HA-immunoprecipitation (Lysosome-IP) in H727 cells expressing TMEM192-3×HA as well as sh GFP or sh LZTR1 . Normalization of the Lysosome fraction using LAMP1 and lysosome wash-out using Vinculin. mTOR, SLC3A2, SLC7A5 and Vinculin (or LAMP1) were detected on the same gel. H Immunofluorescence analysis of H727 cells expressing sh GFP or sh LZTR1 after DMSO- or RMC7977 (10 nM, 24 h)-treated H727 cells using the indicated antibodies. Scale bar 20 µm. Colocalization score for all three proteins is shown as mean ± SEM; n = 4 technical replicates. I PLA was performed on H727 cells expressing sh GFP or sh LZTR1 using antibodies against mTOR and SLC3A2. The number of PLA puncta, indicative of mTOR/SLC3A2 proximity, was quantified per nucleus. Scale bar 30 µm. Data are shown as mean ± SEM, n = 4 technical replicates per group. J PLA was performed on lung tumor sections 10 weeks after post-Cre injection using antibodies against mTOR and SLC3A2. Scale bar 50 µm. The number of PLA puncta, indicative of mTOR/SLC3A2 proximity, was quantified per nucleus. Data are shown as mean ± SEM, n = 4 mice per group, with p -value calculated by two-sided Mann-Whitney test.

Article Snippet: The combinations of antibodies used for PLA on human cells are listed below: On the tumor tissue, a different combination was used: Goat Minus probe (DUO92006) with anti-LAMP1 (Novus Biologicals, AF4320) or SLC3A2 /LAT1 Antibody (Novus Biologicals, NBP1-51935, combined with Rabbit Plus probe (DUO92005) with mTOR (Cell Signaling, #2983).

Techniques: Labeling, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Immunofluorescence, Injection, MANN-WHITNEY

Journal: Amino Acids

Article Title: Targeting LAT1 with JPH203 to reduce TNBC proliferation and reshape suppressive immune microenvironment by blocking essential amino acid uptake

doi: 10.1007/s00726-025-03456-3

Figure Lengend Snippet: Expression patterns of LAT1, LAT2, and 4 F2 hc in breast cancer subtypes. A RNA-seq data from TCGA, METABRIC, and HNCH-BC databases showing the expression levels of LAT1 ( SLC7 A5 ), LAT2 ( SLC7 A8 ), and 4 F2 hc ( SLC3 A2 ) across different breast cancer subtypes. Donut charts indicate the distribution of molecular subtypes in each database, while mountain plots depict the differences in transcript levels (*** p < 0.001). Immunohistochemistry (IHC) demonstrate LAT1 and LAT2 protein levels in tumor tissue samples. B Representative IHC images for LAT1 and LAT2 proteins in breast cancer subtypes ( n = 20/subtype), quantified by average optical density (AOD) (* p < 0.05; ** p < 0.01). C Distribution of LAT1 and LAT2 protein levels across breast cancer subtypes

Article Snippet: The antibodies used included PerCP-conjugated mouse monoclonal anti-LAT1 antibody (#NBP2-50465PCP, Novus Biologicals), unconjugated mouse monoclonal anti-LAT2 antibody (#MA5-24,943, Invitrogen), and Alexa Fluor 488 goat anti-mouse secondary antibody (#A28175, Invitrogen).

Techniques: Expressing, RNA Sequencing, Immunohistochemistry

Journal: Amino Acids

Article Title: Targeting LAT1 with JPH203 to reduce TNBC proliferation and reshape suppressive immune microenvironment by blocking essential amino acid uptake

doi: 10.1007/s00726-025-03456-3

Figure Lengend Snippet: Therapeutic potential of targeting LAT1 in TNBC. A Quantitative analysis of LAT1 and LAT2 transcript levels in five breast cancer cell lines (MDA-MB-231, HCC1937, SK-BR-3, BT474, and T47D) using qRT-PCR, normalized to GAPDH and plotted as fold changes relative to T47D. B Flow cytometry analysis showing LAT1 and LAT2 protein expression on the cell surface of breast cancer cell lines, expressed as mean fluorescence intensity (MFI). C LAT1 knockdown using shRNA (shSLC7 A5-1 or shSLC7 A5-2) in MDA-MB-231 and HCC1937 cells. Flow cytometry validated LAT1 expression levels (Bank: wild type; shCtrl: empty shRNA). Proliferation was assessed using the CCK-8 assay (*** p < 0.001), and EAA levels were analyzed via metabolomics (*** p < 0.001). Data are presented as mean ± SD from three independent experiments

Article Snippet: The antibodies used included PerCP-conjugated mouse monoclonal anti-LAT1 antibody (#NBP2-50465PCP, Novus Biologicals), unconjugated mouse monoclonal anti-LAT2 antibody (#MA5-24,943, Invitrogen), and Alexa Fluor 488 goat anti-mouse secondary antibody (#A28175, Invitrogen).

Techniques: Quantitative RT-PCR, Flow Cytometry, Expressing, Fluorescence, Knockdown, shRNA, CCK-8 Assay

Journal: PLOS Genetics

Article Title: MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis

doi: 10.1371/journal.pgen.1010904

Figure Lengend Snippet: A-C, top panels. U2OS MYC-ER ( A ), SHEP N-MYC-ER, ( B ), and SKNAS N-MYC-ER ( C ) were treated with ethanol control (MYC-OFF) or 4- hydroxytamoxifen (MYC-ON) (4OHT) to activate MYC, and entrained with dexamethasone, and after 24 hours, protein was collected every 4 hours for the indicated time period. Protein lysates were prepared to preserve protein glycosylation (see ), and immunoblot was performed for the indicated proteins. For some targets [4F2hc, LAT1, REV-ERBα (abbreviated REVα)], a darker exposure (‘dark’) and lighter exposure (‘light’) of the same blot are presented. For GLUT1, # indicates a non-specific band. Some samples (CT26 for U2OS, CT32 for SHEP and SKNAS) were treated with PNGase-F prior to immunoblot to remove glycosylation marks. Note that the PNGase-F lanes have less protein loaded than the other lanes. Data represent n = 3–4 biological replicates for each cell line. A-C, bottom panels. Results from n = 3–4 immunoblot replicates were quantified, relative to Tubulin, and analyzed in ECHO for circadian rhythmicity. Displayed curves are the baseline-subtracted and smoothed outputs from ECHO analysis. ECHO norm. = ECHO normalized. Proteins with a 22–26 hour period and a p value < 0.05 and a BH.Adj.P.Value < 0.05 were deemed rhythmic, which is indicated in the Figure. Note the inset MYC-OFF only graphs for U2OS LAT1, SKNAS LAT1, SKNAS 4F2hc, and SKNAS REV-ERBα, which show oscillation of these proteins in MYC-OFF cells on a different scale.

Article Snippet: Wells were washed with tris-buffered saline, and stained with primary antibodies mouse anti-LAT1 BU53 (Novus NBP2-50465AF647) or mouse IgG2A isotype control (Novus IC003R), and secondary antibodies goat anti-mouse Alexa Fluor 790 (Thermo Scientific A11357) or CellTag 700 Stain (Licor 926–41090), which is used to quantify total cell number and intensity.

Techniques: Control, Glycoproteomics, Western Blot

Journal: PLOS Genetics

Article Title: MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis

doi: 10.1371/journal.pgen.1010904

Figure Lengend Snippet: A. On-cell western of U2OS MYC-ER cells ± MYC for LAT1. U2OS MYC-ER cells were grown on a 24-well tissue culture plate ± 4OHT for 48 hours, fixed with formaldehyde but not permeabilized, and then stained with the indicated antibody or IgG control. CellStain 700 indicates cell density in each well. Data represent at least two independent experiments of 6 biological replicate wells each. B. Quantitation of LAT1 or IgG from ( A ). * indicates P < 0.00001 by Welch’s Corrected Student’s T-test. C. LC-Mass spectrometry was performed on U2OS MYC-ER treated ± 4OHT for at least 48 hours. N = 25 circadian timepoints for MYC-OFF and MYC-ON were averaged as biological replicates, normalized to cell number for each collection. * indicates p < 0.05 by Welch’s Corrected Student’s T-test.

Article Snippet: Wells were washed with tris-buffered saline, and stained with primary antibodies mouse anti-LAT1 BU53 (Novus NBP2-50465AF647) or mouse IgG2A isotype control (Novus IC003R), and secondary antibodies goat anti-mouse Alexa Fluor 790 (Thermo Scientific A11357) or CellTag 700 Stain (Licor 926–41090), which is used to quantify total cell number and intensity.

Techniques: Western Blot, Staining, Control, Quantitation Assay, Mass Spectrometry