Journal: bioRxiv

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.1101/255745

Figure Lengend Snippet: ( A ) Schematic diagram showing the stable HEK 293ET cell lines expressing iRhom proteins or related rhomboid pseudoproteases as controls, which were subjected to immunoprecipitation followed by mass spectrometry. ( B ) An example immunoprecipitation from the cell lines indicating that only immunoprecipitates from cell lines expressing WT iRhom1 or iRhom2 contain the binding of the positive control protein, TACE. Here and throughout, immature and mature species of TACE are indicated with white and black arrowheads respectively. The red arrows show the full-length forms of the individual rhomboid-like proteins. ( C ) Peptides assigned to FRMD8 that were found in immunoprecipitates from iRhom1, iRhom2 or the N-terminus of iRhom1 but not in the other samples. The peptides are shown from a representative experiment. ( D ) All of the peptides found in the iRhom immunoprecipitates from (C) were mapped unto the human FRMD8 amino acid sequence. ( E ) Schematic diagram illustrating the domain structure of iTAP/FRMD8. ( F ) Alignment of the central FERM lobe (FERM-M) from iTAP/FRMD8 versus the pfam FERM-M consensus ( pfam00373 ).

Article Snippet: The following antibodies were used: TACE (Ab39162, Abcam); TACE Ab318 (monoclonal anti-TACE antibody ( ) was used for the detection of mouse TACE; alpha-Tubulin (sc-8035, Santa Cruz), alpha-Tubulin (Clone YL1/2, Ana Regaldo, IGC), p97 (Thermo, MA1-21412), HA-HRP (clone 3F10, Roche), HA (clone HA.11, 901501, Biolegend) V5-HRP (R961-25, Life Technologies), Frmd8 (157H00083786-B01P, Abnova), Transferrin Receptor (13-6800, Life Technologies), Flag-HRP (A8592, Sigma), GAPDH (2118, CST), Actin (abcam ab8227).

Techniques: Expressing, Immunoprecipitation, Mass Spectrometry, Binding Assay, Positive Control, Sequencing

Journal: bioRxiv

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.1101/255745

Figure Lengend Snippet: ( A ) Lysates were prepared from a panel of tissues from WT C57 BL/6J mice and iTAP was detected by western blot. Lysates from WT versus iTap KO L929 mouse fibroblasts were used as a control. ( B ) The extent of co-expression between iTap/Frmd8, TACE/ADAM17, iRhom2 or iRhom1 in mouse anatomical parts and cell types was investigated by interrogating gene expression data from the GeneChip™ Mouse Genome 430 2.0 Array via the Genevestigator platform .

Article Snippet: The following antibodies were used: TACE (Ab39162, Abcam); TACE Ab318 (monoclonal anti-TACE antibody ( ) was used for the detection of mouse TACE; alpha-Tubulin (sc-8035, Santa Cruz), alpha-Tubulin (Clone YL1/2, Ana Regaldo, IGC), p97 (Thermo, MA1-21412), HA-HRP (clone 3F10, Roche), HA (clone HA.11, 901501, Biolegend) V5-HRP (R961-25, Life Technologies), Frmd8 (157H00083786-B01P, Abnova), Transferrin Receptor (13-6800, Life Technologies), Flag-HRP (A8592, Sigma), GAPDH (2118, CST), Actin (abcam ab8227).

Techniques: Western Blot, Control, Expressing, Gene Expression

Journal: bioRxiv

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.1101/255745

Figure Lengend Snippet: ( A ). Schematic representation of the CRISPR targeting strategy to delete mouse Frmd8 / iTap gene using two guide RNAs flanking the first coding exon (exon 2). ( B ). Mouse embryonic fibroblasts were isolated from WT versus two independent iTap KO E14.5 embryo littermates. The loss of iTap at the protein level is shown by immunoblotting. ( C ). ConA-enriched lysates from WT versus iTap KO MEFs were deglycosylated as described previously. The transferrin receptor (TfR) is used as a loading control. ( D ). Mature TACE is depleted or diminished in TACE-relevant tissues from iTAP KO mice. ConA-enriched lysates from WT vs iTap KO mouse tissues and bone marrow-derived macrophages, were deglycosylated as described previously. TACE was detected by western blot. The immature and mature species of TACE are indicated with white arrowheads and black arrowheads respectively, whereas red arrowheads denote the fully deglycosylated mature polypeptide. The experiment was performed twice with lysates isolated from tissues from two individual KO mice. ( E ). iTAP is essential for TACE physiological regulation in human primary cells. Isolated primary human peripheral blood mononuclear cells (PBMC) were differentiated into monocytes, then electroporated with the indicated siRNAs. Cells were then stimulated with the indicated concentrations of lipopolysaccharide (LPS). After 18h, the concentration of the cytokines TNF, IL-6 and IL-8 secreted into the supernatants was measured by ELISA. The experiment was done three independent times and data from one representative experiment is shown. Data presented as mean ± standard error from triplicate measurements.

Article Snippet: The following antibodies were used: TACE (Ab39162, Abcam); TACE Ab318 (monoclonal anti-TACE antibody ( ) was used for the detection of mouse TACE; alpha-Tubulin (sc-8035, Santa Cruz), alpha-Tubulin (Clone YL1/2, Ana Regaldo, IGC), p97 (Thermo, MA1-21412), HA-HRP (clone 3F10, Roche), HA (clone HA.11, 901501, Biolegend) V5-HRP (R961-25, Life Technologies), Frmd8 (157H00083786-B01P, Abnova), Transferrin Receptor (13-6800, Life Technologies), Flag-HRP (A8592, Sigma), GAPDH (2118, CST), Actin (abcam ab8227).

Techniques: CRISPR, Isolation, Western Blot, Control, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.1101/255745

Figure Lengend Snippet: A pair of gRNAs (indicated in red) was selected to engineer the deletion of the first coding exon of mouse Frmd8/iTap as described in materials and methods. The PAM sequence and the theoretical Cas9 cut sites 3-4 bp from the PAM are indicated. The resulting founder animals were genotyped from tail biopsies and the identity of the lesions revealed by DNA sequencing. Two lines of animals (A and B) were established following germline transmission of the mutant alleles from individual founders. KO line A contains a 478 bp deletion that removes exon 2 along with parts of introns 1 and 2. KO line B contains a larger deletion of 625 bp that starts within the non-coding exon 1, deletes all of intron 1, exon 2 and part of intron 2, as shown. In both cases, the precise nucleotide sequence on the 5’ and 3’ boundaries of the mutant lines is indicated and aligned with the WT genomic sequence.

Article Snippet: The following antibodies were used: TACE (Ab39162, Abcam); TACE Ab318 (monoclonal anti-TACE antibody ( ) was used for the detection of mouse TACE; alpha-Tubulin (sc-8035, Santa Cruz), alpha-Tubulin (Clone YL1/2, Ana Regaldo, IGC), p97 (Thermo, MA1-21412), HA-HRP (clone 3F10, Roche), HA (clone HA.11, 901501, Biolegend) V5-HRP (R961-25, Life Technologies), Frmd8 (157H00083786-B01P, Abnova), Transferrin Receptor (13-6800, Life Technologies), Flag-HRP (A8592, Sigma), GAPDH (2118, CST), Actin (abcam ab8227).

Techniques: Sequencing, DNA Sequencing, Transmission Assay, Mutagenesis

Journal: Diabetes

Article Title: From Single Nucleotide Polymorphism to Transcriptional Mechanism

doi: 10.2337/db12-1416

Figure Lengend Snippet: Overview of the analysis strategy ( – ) to identify the putative regulatory effect of GWAS candidate on FRMD3 regulation , linking the gene to transcriptional regulation of the BMP pathway ( – ) and DN and suggesting a hypothetical regulatory model .

Article Snippet: We searched for a common module among promoter sequences of a subset of the eight FRMD3 coexpressed BMP pathway members and the SNP-altered sequence of the FRMD3 promoter (FrameWorker, Genomatix).

Techniques:

Journal: Diabetes

Article Title: From Single Nucleotide Polymorphism to Transcriptional Mechanism

doi: 10.2337/db12-1416

Figure Lengend Snippet: Functional association of FRMD3 -correlated genes. Top 10 pathways (Ingenuity Pathways Analysis; Ingenuity Systems) of 581 FRMD3 -correlated genes sorted by the ratio of members of the pathway among FRMD3 -correlated genes vs. total number of members of that pathway. P ≤ 0.001 for all pathways. Junct., junction; med., mediated; Ox., oxidative; Pot., potentiation; Sign., signaling.

Article Snippet: We searched for a common module among promoter sequences of a subset of the eight FRMD3 coexpressed BMP pathway members and the SNP-altered sequence of the FRMD3 promoter (FrameWorker, Genomatix).

Techniques: Functional Assay

Journal: Viruses

Article Title: Identification and Functional Analyses of Host Proteins Interacting with the p17 Protein of Avian Reovirus

doi: 10.3390/v14050892

Figure Lengend Snippet: The positive proteins used for yeast two-hybrid analysis.

Article Snippet: 15 , XM_040694238.1 , Gallus gallus FERM domain containing 8 (FRMD8) , It has confirmed the pathophysiological significance of FRMD8 in iPSC-derived human macrophages and mouse tissues, thus demonstrating its role in the regulated release of multiple cytokine and growth factor signals. , 9815.

Techniques: Expressing, Control, Migration, Coagulation, Scaffolding, Transduction, Activation Assay, Ubiquitin Proteomics, Gene Expression, Cell Differentiation

Journal: Neuropharmacology

Article Title: Effects of a novel phosphodiesterase 10A inhibitor in non-human primates: a therapeutic approach for schizophrenia with improved side effect profile

doi: 10.1016/j.neuropharm.2016.08.012

Figure Lengend Snippet: Plasma concentration of FRM-6308 in monkeys after single drug administration

Article Snippet: FRM-6308 (FORUM Pharmaceuticals, Inc.) is a novel, potent and highly selective PDE10A inhibitor that has also demonstrated antipsychotic efficacy in rodent models (conditioned avoidance responding, stimulant-induced locomotor activity and prepulse inhibition tests).

Techniques: Clinical Proteomics, Concentration Assay

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) A diagram of the Frmd8 targeted alleles. Exons 4, 5, and 6 are flanked by loxp sites. ( B ) Distinguishing the genotype of littermate mice by mice coat color. Yellow represents MMTV-Cre + ; Frmd8 fl/fl ; PyMT genotype, and black represents MMTV-Cre - ; Frmd8 fl/fl ; PyMT genotype. ( C ) Representative PCR genotyping of mouse tail DNA. ( D ) Relative mRNA level of Frmd8 in 7-week-old mammary glands from PyMT mice was analyzed by qRT-PCR. Gapdh was used as an internal reference. *p<0.05 by unpaired Student’s t -test, n = 3. ( E ) Kaplan–Meier plot showing the appearance of palpable tumors in MMTV-Cre - ; Frmd8 fl/fl ; PyMT (n = 9) and MMTV-Cre + ; Frmd8 fl/fl ; PyMT (n = 9) mice (Log-rank test). ( F ) Representative images of tumors from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. ( G, H ) Total tumor weight ( G ) and number of tumors ( H ) per mice were measured. **p<0.01, ***p<0.001 by unpaired Student’s t -test. ( I ) Representative H&E staining of tumors from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. Scale bar, 50 μm. ( J ) Immunohistochemistry (IHC) staining for Ki67 expression in mammary tumors from PyMT mice. The black boxes represent the magnified typical staining of the original images. Scale bar, 50 μm. ( K ) Quantification of Ki67-positive cell percentage in ( J ). *p<0.05 by unpaired Student’s t -test, n = 3. ( L ) Kaplan–Meier plot showing the appearance of palpable tumors in MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice, with or without tamoxifen treatment (Log-rank test, *p<0.05, ***p<0.001, ****p<0.0001). ( M ) Representative images of tumors from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice, with or without tamoxifen treatment. ( N, O ) Total tumor weight ( N ) and number of tumors ( O ) per mice from ( L ) were measured (n ≥ 10). *p<0.05, **p<0.01, ***p<0.001 by unpaired Student’s t -test or Mann–Whitney test. Figure 1—source data 1. Unedited gel pictures for , indicating the relevant bands. Figure 1—source data 2. Original files for gel pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Quantitative RT-PCR, Staining, Immunohistochemistry, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) Immunohistochemistry staining for Frmd8 expression in mammary glands from PyMT mice. The black boxes represent the magnified typical staining of the original images. Scale bar, 50 μm. ( B ) Lysates from different tissues of MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice were examined by western blot. Specific bands are marked with an arrow. Figure 1—figure supplement 1—source data 1. Unedited western blot pictures for , indicating the relevant bands. Figure 1—figure supplement 1—source data 2. Original files for western blot pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) T-SNE plot showing the distribution of cells from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. ( B ) T-SNE plot showing the distribution of epithelial, immune, and stromal cells. ( C, D ) T-SNE plot showing the distribution of main cell lineages ( C ) and their relative percentage ( D ). ( E, F ) T-SNE plot showing the distribution of epithelial cell lineages ( E ) and their relative percentage ( F ). ( G ) Heatmap showing distinct features of each cell lineages. Rows, genes. Columns, cells. The color key from blue to red indicates low to high gene expression. ( H ) Boxplot showing copy number variation (CNV) score of main cell lineages. ( I ) Dot plot showing the expression of Esr1 and Pgr in epithelial cell lineages. ( J ) T-SNE plot showing the distribution of normal cells and tumor cells. ( K ) Dot plot showing the expression of Frmd8 in normal and tumor cells from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. ( L ) Statistical analysis of Frmd8 , Esr1 and Pgr expression in normal and tumor cells from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. *p<0.05, ****p<0.0001 by Mann–Whitney test.

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Gene Expression, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) Dot plot representing the expression level (red jet) and the number of expressing cells (dot size) of the top markers from main cell lineages. ( B ) T-SNE visualization of the expression level of main lineages markers. The color key from gray to pink indicates low to high gene expression. ( C ) T-SNE visualization of the expression level of the top markers from each of the cell lineages. The color key from gray to blue indicates low to high gene expression. ( D ) Dot plot representing the expression level (red jet) and the number of expressing cells (dot size) of the top markers from each of the cell lineages. ( E ) Dot plot showing the expression of Esr1 and Pgr in normal and tumor cells from MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice.

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Expressing, Gene Expression

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) Representative multiplex immunofluorescence images of tumor tissues and tissues adjacent to tumor from MMTV-Cre - ; Frmd8 fl/f l ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. Scale bar, 50 μm. ( B ) Qualification of Frmd8 and CK8 expression (left panel) and ERα and PR-positive cell percentage in CK8 + cells (right panel) in ( A ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA (left panel) or unpaired Student’s t -test (right panel), n ≥ 3. ( C ) Lysates from MCF7 cells transiently transfected with Flag or Flag-FRMD8 were immunoblotted. ( D ) Lysates from MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA were immunoblotted. In this and subsequent figures, specific bands are marked with an arrow. ( E ) Relative mRNA levels of ESR1 and FRMD8 from MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA were analyzed by qRT-PCR. GAPDH was used as an internal reference. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, n = 2. ( F ) Lysates from MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA were immunoblotted. ( G ) Lysates from MCF7 and T47D cells transiently transfected with HA or HA-FRMD8 were immunoblotted. ( H, I ) Lysates of MCF7 cells co-transfected with HA-FRMD8 and FOXO3A siRNA as indicated were immunoblotted ( H ). ERα protein levels were quantified by normalizing to the intensity of the GAPDH band ( I ). **p<0.01 by unpaired Student’s t -test, n = 3. ( J ) Lysates of T47D cells transfected with control or FRMD8 siRNA were subjected to anti-FOXO3A ChIP-qPCR. *p<0.05, ***p<0.001 by one-way ANOVA, n = 2. ( K ) Working model for FRMD8 promotes ESR1 transcription via upregulating FOXO3A expression. Figure 3—source data 1. Unedited western blot pictures for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Multiplex Assay, Immunofluorescence, Expressing, Transfection, Control, Quantitative RT-PCR, ChIP-qPCR, Western Blot

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A–D ) The scatter plot shows the cells in multiplex immunofluorescence images of normal tissues adjacent to tumor in MMTV-Cre - ; Frmd8 fl/fl ; PyMT and MMTV-Cre + ; Frmd8 fl/fl ; PyMT mice. ( E ) Immunohistochemistry (IHC) staining for Frmd8 and ERα expression in 7-week-old mammary glands from PyMT mice. The black boxes represent the magnified typical staining of the original images. Scale bar, 100 μm.

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Multiplex Assay, Immunofluorescence, Immunohistochemistry, Expressing, Staining

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) Lysates of HEK293T cells co-transfected with Flag-ERα and FRMD8 siRNA as indicated were immunoblotted. ( B, C ) Lysates from T47D cells transiently transfected with control or FRMD8 siRNA were subjected to immunoblotting. Cells were treated with 100 μg/ml CHX for the indicated times ( B ). ERα protein levels were quantified by normalizing to the intensity of the Actin band, n = 4 ( C ). ( D ) Immunoblot analysis of ERα in MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA. The cells were treated with MG132 (25 μM) or chloroquine (50 μM) for 6 h. Figure 4—source data 1. Unedited western blot pictures for , indicating the relevant bands. Figure 4—source data 2. Original files for western blot pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Transfection, Control, Western Blot

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A, B ) Lysates of MCF7 ( A ) and T47D ( B ) cells transfected with Flag or Flag-FRMD8 were anti-Flag immunoprecipitated and immunoblotted for ERα and Flag. ( C ) Lysates from MCF7 cells were immunoprecipitated with IgG or anti-ERα, then immunoblotted for FRMD8 and ERα. ( D ) HEK293T cells were transiently transfected with Flag-ERα. ERα proteins in HEK293T whole-cell lysates (WCL) pulled down by GST or GST-FRMD8 recombinant proteins were subjected to western blot. Asterisks indicate proteins at the expected molecular weight. ( E ) Venn diagram showing overlap of E3 ligases related to ERα and proteins interacting with FRMD8. ( F ) Lysates of HEK293T cells transfected with Flag or Flag-FRMD8 were anti-Flag immunoprecipitated and immunoblotted. ( G ) Lysates of HEK293T cells transfected with Flag or Flag-UBE3A were anti-Flag immunoprecipitated and immunoblotted. ( H, I ) HEK293T ( H ) and T47D ( I ) cells were co-transfected with Flag-ERα, HA-Ub, and FRMD8 siRNA as indicated. Cells were treated with MG132 (25 μM) for 6 h. WCL were immunoprecipitated with anti-Flag and then immunoblotted for ubiquitinated ERα. ( J, K ) Lysates of T47D cells co-transfected with FRMD8 and UBE3A siRNA as indicated were immunoblotted ( J ). ERα protein levels were quantified by normalizing to the intensity of the Actin band ( K ). ***p<0.001, ****p<0.0001 by one-way ANOVA, n = 3. ( L ) Working model for FRMD8 disrupts the interaction between ERα and UBE3A, and protects ERα from UBE3A-mediated degradation. Figure 5—source data 1. Unedited western blot pictures for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Transfection, Immunoprecipitation, Recombinant, Western Blot, Molecular Weight

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) Dot plot showing the expression of Frmd8 in epithelial cell lineages from PyMT mice. ( B ) Lysates from human mammary epithelial cell (MEC) and breast cancer cells were subjected to immunoblotting. ( C ) Methylation of FRMD8 promoter region in breast cancer according to the University of California Santa Cruz (UCSC) database ( http://xena.ucsc.edu/ ). ( D ) SUM159 and MDA-MB-231 cells were treated with 5-Aza-dC (10 μM) for 48 h. Protein expression of FRMD8 was examined by western blot. ( E ) SUM159 and MDA-MB-231 cells were treated with 5-Aza-dC (10 μM) for 48 h. FRMD8 mRNA levels was examined by quantitative reverse transcription PCR (qRT-PCR) GAPDH was used as an internal reference. **p<0.01 by unpaired Student’s t -test, n = 3. ( F ) IHC analysis of FRMD8 expression in human breast carcinoma TMA was performed. Representative examples (scale bar, 100 μm) of normal tissue adjacent to tumor and breast cancer with different levels of FRMD8 expression are shown, with the magnification of selected areas inserted. ( G ) Kaplan–Meier analysis for the overall survival of breast cancer patients according to FRMD8 expression (Log-rank test). ( H–J ) Recurrence-free survival of breast cancer patients according to FRMD8 expression were analyzed according to Kaplan–Meier plotter ( http://kmplot.com/analysis/ ). Figure 6—source data 1. Unedited western blot pictures for , indicating the relevant bands. Figure 6—source data 2. Original files for western blot pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Expressing, Western Blot, Methylation, Reverse Transcription, Quantitative RT-PCR

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: ( A ) MCF7 and T47D cells were treated with 5-Aza-dC (10 μM) for 48 h. Protein expression of FRMD8 was examined by western blot. ( B, C ) Recurrence-free survival ( B ) and overall survival ( C ) of breast cancer patients with tamoxifen treatment according to FRMD8 expression were analyzed according to Kaplan–Meier plotter. Figure 6—figure supplement 1—source data 1. Unedited western blot pictures for , indicating the relevant bands. Figure 6—figure supplement 1—source data 2. Original files for western blot pictures displayed in .

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Loss function of tumor suppressor FRMD8 confers resistance to tamoxifen therapy via a dual mechanism

doi: 10.7554/eLife.101888

Figure Lengend Snippet: A working model shows that loss of FRMD8 promotes mammary tumor progression and confers tamoxifen resistance by downregulating ERα expression at both transcriptional and post-translational levels.

Article Snippet: Anti-FRMD8 (#HPA002861) antibody was from Atlas Antibodies.

Techniques: Expressing