antibodies against pe foxo1  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: Epinephrine promotes breast cancer metastasis through a ubiquitin-specific peptidase 22–mediated lipolysis circuit

doi: 10.1126/sciadv.ado1533

Figure Lengend Snippet: ( A to F ) ATGL expression in breast cancer tissues by IHC staining. Representative images from para-tumor normal tissue and cancer sections (A) and quantification from 65 patients (B) are shown. Scale bars, 50 μm. The data were further analyzed by EPI levels [(C) and (D)]. The correlation of ATGL with EPI (E) and USP22 (F) was analyzed. ( G to J ) The mRNA [(G) and (H)] and protein [(I) and (J)] levels of ATGL in MDA-MB-231 cells treated with EPI (10 nM) were determined by real-time PCR and Western blotting, respectively. ( K to M ) Intracellular lipid analysis, including BODIPY [(K) and (L)], DAG (M), and FFAs (N) in WT and USP22-null MDA-MB-231 cells treated with EPI in vitro. ( O to R ) Intracellular lipid analysis in WT and USP22-null MDA-MB-231 cells isolated from the xenograft tumors as shown in . ( S ) The protein levels of USP22, FOXO1, and ATGL in the WT 4T1 cells, USP22 KO 4T1 cells, and the USP22 KO 4T1 cells transduced with ATGL were measured. ( T to Za ) A total of 1 × 10 5 WT, USP22 KO 4T1 cells, or USP22 KO 4T1 cells transduced with ATGL were injected into the mammary fat pad of BALB/c mice. Tumor volume (T), bioluminescence activity [(U) and (V)], and tumor weight [(W) and (X)] were measured. Lung metastases were measured by luminol fluorescence [(U) and (Y)] and H&E staining of their lung sections [(Z) and (Za)]. N = 5 each group. Pearson correlation coefficient was used as a measure of association. Statistical significance was determined by one-way ANOVA test or unpaired Student’s t test. Data are expressed as means ± SD of five independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no statistical significance.

Article Snippet: The cells were then stained with specific antibodies against PE-FoxO1 (14262, Cell Signaling Technology) for 1 hour at 4°C.

Techniques: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot, In Vitro, Isolation, Transduction, Injection, Activity Assay, Fluorescence, Staining

Journal: Science Advances

Article Title: Epinephrine promotes breast cancer metastasis through a ubiquitin-specific peptidase 22–mediated lipolysis circuit

doi: 10.1126/sciadv.ado1533

Figure Lengend Snippet: ( A to E ) Effect of EPI on FOXO1 protein expression in WT and USP22-null MDA-MB-231 cells without [(A) and (B)] or with MG132 treatment [(C) and (D)]. The mRNA levels of FOXO1 were analyzed by real-time RT-PCR (E). ( F to I ) Analysis of USP22, FOXO1, and ATGL expression levels by IHC in syngeneic tumor tissues as shown in . Representative images (F) (scale bars, 50 μm) and quantification data [(G) to (I)] are shown. ( J to M ) FOXO1 interaction in HEK239T cells transiently transfected with FOXO1 and USP22 (J) or USP22 truncated mutants (K) expressing plasmids and in MDA-MB-231 cells (L) or further with EPI treatment (M). WB, Western blotting. WCL, whole cell lysate. ( N to P ) Analysis of FOXO1 ubiquitination in HEK293T cells coexpressed with USP22 (N) and its mutants (O) and in USP22 WT and KO MDA-MB-231 cells (P). ( Q ) Immunoblot analysis of FOXO1 protein degradation in HEK293T cells transfected with or without USP22 ( n = 3). ( R ) Analysis of FOXO1 binding to Atgl promoter by ChIP in WT and USP22 KO MDA-MB-231 cells ( n = 5). ( S ) Reconstitution of FOXO1 expression in USP22-null MDA-MB-231 cells restored ATGL expression. ( T to Y ) Analysis of FOXO1 expression in human breast cancer tissues [(T) and (U) with para-tumor tissues as controls or (V) and (W) in EPI high and EPI low groups] as well as its correlation with USP22 (X) and EPI (Y). Scale bars, 50 μm. Pearson correlation coefficient was used as a measure of association. Statistical significance was determined by one-way ANOVA test or unpaired Student’s t test. Data are expressed as means ± SD of three or more independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ****P < 0.0001. ns, no statistical significance.

Article Snippet: The cells were then stained with specific antibodies against PE-FoxO1 (14262, Cell Signaling Technology) for 1 hour at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Ubiquitin Proteomics, Binding Assay

Journal: Science Advances

Article Title: Epinephrine promotes breast cancer metastasis through a ubiquitin-specific peptidase 22–mediated lipolysis circuit

doi: 10.1126/sciadv.ado1533

Figure Lengend Snippet: ( A and B ) Proteomic analysis of USP22 phosphorylation from HEK293T cells transfected with or without Flag-AKT. The spectra of peptides carrying phosphorylated peptides (A) and the identified phosphorylation residues of USP22 (top) and AKT (bottom) (B) are shown. ( C and D ) Analysis of phosphorylation (C) and ubiquitination (D) with USP22 and its phosphorylation mutant. ( E ) Analysis of FOXO1 interaction with USP22 and its phosphorylation mutant. ( F and G ) Analysis of protein stability of USP22 and its phosphorylation mutant ( n = 3). ( H to O ) Analysis of USP22 phosphorylation in human breast cancer by IHC. Representative images in tumor and para-controls (H) or in EPI high and EPI low groups (J) as well as the quantification from 65 patients [(I) and (K)] are shown. Scale bars, 50 μm. Statistical analyses of the correlation p-USP22-Thr 147 levels with EPI (L), total USP22 (M), FOXO1 (N), and ATGL (O) are shown. Pearson correlation coefficient was used as a measure of association. Statistical significance was determined by unpaired Student’s t test. Data are expressed as means ± SD of three or more independent experiments. ** P < 0.01; **** P < 0.0001; ns, no statistical significance.

Article Snippet: The cells were then stained with specific antibodies against PE-FoxO1 (14262, Cell Signaling Technology) for 1 hour at 4°C.

Techniques: Phospho-proteomics, Transfection, Ubiquitin Proteomics, Mutagenesis

Journal: Science Advances

Article Title: Epinephrine promotes breast cancer metastasis through a ubiquitin-specific peptidase 22–mediated lipolysis circuit

doi: 10.1126/sciadv.ado1533

Figure Lengend Snippet: ( A to E ) 4T1 TNBC cells were injected into the mammary fat pad of BALB/c mice ( N = 5 each group). Tumor volume (A), bioluminescence activity [(B) and (C)], and tumor weight [(D) and (E)] were measured. ( F to H ) The metastases of the lung were measured by luminol fluorescence [(B) and (H)] and H&E staining [(F) and (G)]. Lung tissue sections were analyzed by H&E staining [(F) and (G)]. Representative lung metastasis (F) and quantifications of five mice each group (G) are shown. ( I to L ) Lipid analysis of syngeneic tumors from (D) by lipid BODIPY [(I) and (J)], DAG (K), and FFAs (L). MFI, mean fluorescence intensity. ( M to O ) IHC analysis of USP22, FOXO1, and ATGL expression levels in syngeneic tumor tissue sections from (D). Quantification data of all five mice each group [(M) to (O)] are shown. (P) Model of targeting chronic stress–mediated cancer metastasis and lipolysis by USP22. Statistical significance was determined by one-way ANOVA test or unpaired Student’s t test. Data are expressed as means ± SD of five independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ****P < 0.0001; ns, no statistical significance.

Article Snippet: The cells were then stained with specific antibodies against PE-FoxO1 (14262, Cell Signaling Technology) for 1 hour at 4°C.

Techniques: Injection, Activity Assay, Fluorescence, Staining, Expressing

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet: Analysis of critical transcription factors in wild-type (WT) and Mb1-cre ABCB7 cKO Fr. C cells (B220 + CD19 + CD43 + BP-1 + ). ( A–G ) Intracellular flow cytometry analysis of EBF1 ( A ), E47 (E2A) ( B ), FOXO1 ( C ), PAX5 ( D ), IKAROS ( E ), AIOLOS ( F ), and IRF4 ( G ) expression. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 6–10 mice/group). ( H, I ) Flow cytometry analysis of CD2 ( H ) and CD25 ( I ) expression. Indicated values are the proportion of Fr. C cells positive for either marker, and quantifications are shown on the right of each plot. Offset histograms are representative of three independent experiments (total of five mice/group). ( J ) Intracellular flow cytometry analysis of TdT expression in Fr. B and Fr. C cells. Indicated values are the proportion of cells positive for TdT expression, and quantifications are shown on the right. Offset histograms are representative of three independent experiments (total of 5–7 mice/group). ( K ) Quantitative real-time PCR analysis of Rag1 and Rag2 expression in sorted Fr. B and Fr. C cells. 18S rRNA was used as an endogenous control, and relative expression values were normalized to expression in WT Fr. B cells. Results were obtained from three independent experiments (total of 3–4 mice/group). ( A–K ) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t -test.

Article Snippet: Antibody , Anti-FOXO1 PE (Rabbit monoclonal, C29H4) , Cell Signaling Technology , Cat#:14262S; RRID: AB_2798437 , FC (1:50).

Techniques: Flow Cytometry, Expressing, Marker, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-FOXO1 PE (Rabbit monoclonal, C29H4) , Cell Signaling Technology , Cat#:14262S; RRID: AB_2798437 , FC (1:50).

Techniques: Transgenic Assay, Selection, Concentration Assay, Sequencing, Recombinant, Flow Cytometry, EdU Assay, DNA Purification, Isolation, Colony-forming Unit Assay, Cell Cycle Assay, Software