anti mta1 antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Fibrillarin shapes oncogenic protein pools and ribosomal composition in triple-negative breast cancer

doi: 10.1101/2025.03.21.644506

Figure Lengend Snippet: ( A ) Violin plot illustrating the distribution of proteins detected in total proteomics based on the Log 2 (FC) compared to control for both KDs after 48 and 96 h of FBL depletion. ( B ) Venn diagram displaying the overlap of downregulated proteins at 48 h and 96 h in both FBL KDs. ( C ) Common enriched biological processes associated with downregulated proteins upon FBL KDs. ( D ) Venn diagram of the overlap between the commonly downregulated proteins, and the transcripts with downregulated translation efficiency. Highlighted in red are proteins with an already described role in breast cancer. ( E - G ) mRNA percentage distribution among the 14 fractions collected by polysome profiling for ( E ) MTA1 , ( F ) IRAK1 , and ( G ) TMSB10 in control and FBL-depleted cells. Ribosomal subunits and 80S fractions are highlighted in light grey, and polysome fractions are highlighted in blue. ( H ) Representative Western Blot images displaying the expression of FBL, MTA1, IRAK1, and TMSB10 in control and FBL-depleted (KD1 and KD2) MDA-MB-231 cells. β-ACTIN serves as a loading control. ( I ) RT-qPCR analysis of MTA1 , IRAK1, and TMSB10 levels from control and FBL-depleted (KD1 and KD2) MDA-MB-231 cells. mRNA expression levels were normalized to GAPDH and are presented relative to the control. ( J ) Representative Western Blot images displaying the expression of FBL, MTA1, IRAK1, and TMSB10 in control and FBL-depleted (KD1 and KD2) hTERT-HME1 cells. β-ACTIN serves as a loading control. Statistical analysis: All data are presented as the mean ± SD, n = 3. ( A ) Welch’s two-tailed t-test; **** P < 0.0001.( I ) Unpaired two-tailed t-test, * P < 0.05, ** P < 0.01, **** P < 0.0001. The cut-off applied to proteomic data analysis was Log 2 (FC) ± 0.585, adj P < 0.05.

Article Snippet: The following antibodies were used at the indicated concentrations: anti-FBL (A303-891A, Bethyl Laboratories, 1:2,500 dilution), anti-β-actin (A5441, Sigma, 1:10,000 dilution), anti-metastasis-associated 1 gene (MTA1) (A300-911A-T, Bethyl Laboratories, 1:2,000), anti-interleukin 1 receptor-associated kinase 1 (IRAK1) (ab302554, Abcam, 1:2,000 dilution), anti-thymosin B10 (sc-514309, Santa Cruz Biotechnology, 1:1,000 dilution), anti-RPS28 (14796-1-AP, Proteintech, 1:2,000 dilution), and anti-RPL22 (25002-1-AP, Proteintech, 1:2,000).

Techniques: Control, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Fibrillarin shapes oncogenic protein pools and ribosomal composition in triple-negative breast cancer

doi: 10.1101/2025.03.21.644506

Figure Lengend Snippet: ( A ) MTT assay of control and RPS28-depleted (KD1 and KD2) cells over a 3-day period (24, 48, and 72 h) upon puromycin selection. ( B ) Crystal violet staining of control and RPS28-depleted (KD1 and KD2) cells, performed 10 days after puromycin selection. ( C ) Representative images from the wound-healing assay showing the wound-healing capacity of control and FBL-depleted (KD1 and KD2) cells upon scratch generation at 0 and 24 h. ( D ) Quantification of wound-healing capacity in RPS28-depleted (KD1 and KD2) cells relative to control. Scale bar: 100 µm. ( E ) Representative images showing cells that migrated through the trans-well in Scr and RPS28-depleted (KD1 and KD2) conditions. Scale bar: 100 µm. ( F ) Quantification of the migration capacity of RPS28-depleted (KD1 and KD2) cells relative to control. ( G ) Polysome profiling for control and RPS28-depleted (KD1 and KD2) cells in a 15-45% sucrose gradient. Peaks for 40S, 60S, 80S, and polysomes are highlighted and correspond to absorbance measurements at 260 nm. ( H - J ) mRNA percentage distribution among the 14 fractions collected by polysome profiling for ( H ) MTA1 , ( I ) IRAK1 , and ( J ) TMSB10 in control and RPS28-depleted cells. The fractions were combined as sub-polysomal (1 to 6) and polysomal (7 to 14). ( K ) Representative Western Blot showing RPS28, MTA1, IRAK1, TMSB10 protein expression in Scr control and RPS28-depleted (KD1 and KD2) MDA-MB-231 cells. β-ACTIN serves as a loading control. ( L) H-score quantification of RPS28 in TNBCs from SCAN-B project. Q1 and Q4, first and fourth quartiles, respectively. ( M ) Representative IHC images showing RPS28, IRAK1 and TMSB10 protein levels (low and high, respectively) in the same patient sample. Scale bar: 40µm. ( N and O ). Violin plots displaying the correlation of RPS28 and ( N ) IRAK1 and ( O ) TMSB10 H-scores in TNBC tumors from the SCAN-B project. ( P and Q ) Violin plots displaying the correlation of RPS28 H-score and ( P ) IRAK1 and ( Q ) TMSB10 mRNAs. Statistical analysis: All experimental data are presented as the mean ± SD, n = 3. ( A, D, and F ) Unpaired two-tailed t-test, * P < 0.05, ** P < 0.01, **** P < 0.0001. ( H-J ) Two-way ANOVA with Dunnett’s multiple comparisons test * adj P < 0.05, ** adj P < 0.01, ns = non-significant. ( N-Q ). Unpaired two-tailed t-test corrected with the BH method for multiple comparisons, * adj P < 0.05, ** adj P < 0.01, *** adj P < 0.001, **** adj P < 0.0001, ns adj P > 0.05.

Article Snippet: The following antibodies were used at the indicated concentrations: anti-FBL (A303-891A, Bethyl Laboratories, 1:2,500 dilution), anti-β-actin (A5441, Sigma, 1:10,000 dilution), anti-metastasis-associated 1 gene (MTA1) (A300-911A-T, Bethyl Laboratories, 1:2,000), anti-interleukin 1 receptor-associated kinase 1 (IRAK1) (ab302554, Abcam, 1:2,000 dilution), anti-thymosin B10 (sc-514309, Santa Cruz Biotechnology, 1:1,000 dilution), anti-RPS28 (14796-1-AP, Proteintech, 1:2,000 dilution), and anti-RPL22 (25002-1-AP, Proteintech, 1:2,000).

Techniques: MTT Assay, Control, Selection, Staining, Wound Healing Assay, Migration, Western Blot, Expressing, Two Tailed Test

Journal: Med (New York, N.Y.)

Article Title: Inactivation of TACC2 epigenetically represses CDKN1A and confers sensitivity to CDK inhibitors.

doi: 10.1016/j.medj.2024.12.002

Figure Lengend Snippet: Figure 3. TACC2 interferes with the NuRD and CoREST corepressor complexes (A) SDS-PAGE and silver staining analysis of proteins immunoprecipitated using an anti-GFP antibody from EGFP-TACC2- or EGFP-overexpressing cells (left). The major interacting proteins are indicated on the right. (B) Schematic of N-terminal EGFP-tagged full-length TACC2 (FL) and the indicated deletion mutants (top) and IP coupled with immunoblot analysis of HEK293T cells transfected with the indicated constructs using the indicated antibodies (bottom). (C) IP of the endogenous TACC2 in ECA109 and KYSE30 cells followed by WB assays using the indicated proteins. (D) HEK293T cells were cotransfected with 3 mg each of vectors expressing FLAG-MTA1 and Myc-MBD3 or expressing FLAG-HMG20B and Myc-LSD1 together with 0, 3, and 6 mg of the vector expressing EGFP-TACC2 for 36 h. IP and WB were then performed with the indicated antibodies. (E) ECA109 cells were transfected with two TACC2 siRNAs and siNC for 48 h. IP and WB were performed with the indicated antibodies. (F) Representative immunofluorescence images of endogenous TACC2, MTA1, MBD3, or HMG20B in ECA109 cells. Green, TACC2; red, MTA1, MBD3, and HMG20B; blue, nuclei. Scale bars, 20 mm. (G) Representative immunofluorescence images of ECA109 cells transfected with TACC2 siRNAs or siNC for 48 h and stained with the indicated antibodies (left) and quantification of the nuclear localization percentage of MTA1, MBD3, or HMG20B (right). Scale bars, 20 mm. At least 100 cells were analyzed. Data are the mean ± SD of 3 independent experiments. Significance was measured by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (H) Cytoplasmic and nuclear proteins from ECA109 cells transfected with TACC2 siRNAs or siNC for 48 h were separated and subjected to WB. (I) IHC analyses were performed with the indicated antibodies on serial sections of ESCC specimens (n = 45). Representative images are shown (left). Scale bars, 200 mm. Correlation between TACC2 levels and the cytoplasm localization of indicated proteins was determined (right). Two-tailed chi-square test; ***p < 0.001. See also Figure S5 and Table S3.

Article Snippet: After the retrieval solution had cooled to room temperature, sections were incubated with diluted anti-TACC2 antibody (rabbit polyclonal, 1:200; 11407-1-AP; Proteintech), anti-Ki67 antibody (rabbit polyclonal, 1:1000; 27309-1-AP; Proteintech), anti-Ki67 antibody (rabbit monoclonal, 1:200; #12202; CST), antiCD3 antibody (rabbit monoclonal, 1:100; ab5690; Abcam), anti-CD11b antibody (rabbit monoclonal, 1:100; ab133357; Abcam), anti-p21 antibody (rabbit monoclonal, 1:200; #2947; CST), anti-MTA1 antibody (rabbit monoclonal, 1:200; #5646; CST), antiMBD3 antibody (rabbit polyclonal, 1:200; 14258-1-AP; Proteintech), anti-HMG20B antibody (rabbit polyclonal, 1:200; 14582-1- AP; Proteintech), overnight at 4 C. Sections were then washed three times with phosphate-buffered saline containing Tween 20 (PBST; ZSGB-BIO) and incubated with goat anti-rabbit peroxidase-conjugated secondary antibodies (DAKO, Santa Clara) for 30min at 37 C. Sections were thenwashed three timeswith PBST and stainedwith 3,30-diaminobenzidine (DAB) for 2min to visualize the target protein.

Techniques: SDS Page, Silver Staining, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Plasmid Preparation, Staining, Two Tailed Test