Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined functional genome survey of therapeutic targets for hepatocellular carcinoma.

doi: 10.1158/1078-0432.CCR-09-2214

Figure Lengend Snippet: Fig. 5. Protein expression in HCC. Hematoxylin and eosin (HE) staining (original magnification, × 100) and immunoperoxidase staining (original magnifications, × 100 and × 400) of AKR1B10, HCAP-G, RRM2, and TPX2 proteins in HCC and adjacent nontumorous liver tissue. The specificity of antibodies was determined by immunoblotting of the KIM-1 cell lysate (left). N, nontumorous liver.

Article Snippet: Anti-TPX2 antibody was purchased from Novus Biologicals.

Techniques: Expressing, Staining, Immunoperoxidase Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

doi: 10.1074/jbc.m114.612903

Figure Lengend Snippet: FIGURE 1. Binding of TPX2 and TPX2-710 to microtubules. A, schematic diagram of the TPX2 constructs (left) and Coomassie Brilliant Blue-stained gel of the purified proteins (right). B, co-sedimentation of TPX2 with microtubules. S, supernatant; P, pellet. The concentration of microtubules in each pair of lanes is noted above. Western blots were stained for TPX2 or tubulin. C, quantification of apparent affinity was performed using a quadratic fit. The experiment was performed twice, and the values were averaged. Error bars, S.D.

Article Snippet: The proteins were then transferred to a PVDF membrane and probed using antibodies against TPX2 (Novus Biologicals, Littleton, CO) and tubulin (DM1A, SigmaAldrich).

Techniques: Binding Assay, Construct, Staining, Purification, Sedimentation, Concentration Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

doi: 10.1074/jbc.m114.612903

Figure Lengend Snippet: FIGURE 2. Binding Dynamics of TPX2 and TPX2-710. A, box plot showing release of TPX2 and TPX2–710 from microtubules in the presence of the indicated concentration of KCl added to the buffer. TPX2 fluorescence is reported as arbitrary units (A.U.). Whiskers define the range, boxes encompass the 25th to 75th quartiles, and lines depict the medians. B, TPX2 and TPX2-710 binding to untreated and subtilisin A-digested microtubules; top panels, fluorescence images of TPX2-Halo or TPX2–710-Halo bound to untreated and subtilisin A-digested microtubules; middle, quantification of TPX2 fluorescence; bottom, polyacrylamide gel showing digested and control microtubules. TPX2 fluorescence was measured for at least 60 microtubules for each of two independent experiments; error bars, S.D. C, kymograph of TPX2-Halo and TPX2-710-Halo on microtubules. Vertical scale bar (time), 60 s; horizontal scale bar, 2 m.

Article Snippet: The proteins were then transferred to a PVDF membrane and probed using antibodies against TPX2 (Novus Biologicals, Littleton, CO) and tubulin (DM1A, SigmaAldrich).

Techniques: Binding Assay, Concentration Assay, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

doi: 10.1074/jbc.m114.612903

Figure Lengend Snippet: FIGURE 4. Inhibition of Eg5 by TPX2 requires both binding to the microtubule and an interaction between TPX2 and Eg5. A, kymographs of Eg5-EGFP before andfollowingtheadditionofTPX2orTPX2–710;arrowhead,timeoftheTPX2addition.B,quantificationofEg5-EGFPvelocity;errorbars,S.D.C,kymographofkinesin-1 EGFP dimers walking on microtubules before and after the addition of TPX2 (arrowhead). 1 nM kinesin-1 EGFP (green) and 500 nM TPX2-Halo (red) were used. D, kymographs of Eg5-EGFP (green) before and following the addition of 20 nM TPX2-Halo (red). Right panels, enlarged view. E, kymographs of Eg5-EGFP that was premixedwithTPX2-HaloorTPX2–710-Halo.F,quantificationofEg5-EGFPvelocityinthepresenceof50nMTPX2thatwasHalo-tagged(left)oruntagged(right).Error bars, S.E. Horizontal scale bars (A, C, and E), 1 m; horizontal scale bar (D), 2 m; vertical scale bar, 60 s (A, D, and E) and 5 s (C).

Article Snippet: The proteins were then transferred to a PVDF membrane and probed using antibodies against TPX2 (Novus Biologicals, Littleton, CO) and tubulin (DM1A, SigmaAldrich).

Techniques: Inhibition, Binding Assay

Journal: Oncology letters

Article Title: Reversal of the tamoxifen‑resistant breast cancer malignant phenotype by proliferation inhibition with bromosulfonamidine amino‑podophyllotoxin.

doi: 10.3892/ol.2024.14506

Figure Lengend Snippet: Figure 4. Effect of BSAPPT on apoptosis and cycle‑related gene or protein expression in MCF‑7, MCF7/TAMR and other cancer cells. (A) mRNA expres‑ sion levels of genes linked to the cell cycle and apoptosis were measured by qPCR both before and after MCF‑7 and MCF7/TAMR cells were treated with 10 µg/ml BSAPPT. (B) Western blotting detection of apoptotic and cycle‑related protein expression variations in MCF‑7 and MCF7/TAMR cells before and after using 10 µg/ml BSAPPT. Results of qPCR analysis that assessed differences in the level of expression of genes linked to the cell cycle and apoptosis before and after (C) A549 and (D) MDA‑MB‑231 cells were treated with 10 µg/ml BSAPPT. *P<0.05; **P<0.01; ***P<0.001. BSAPPT, bromosulfonamidine amino‑podophyllotoxin; qPCR, quantitative PCR; Bcl‑2, B‑cell lymphoma 2; Caspase, cysteine aspartic acid‑specific protease; PLK, polo like kinase; CCNB1, cyclin B1; TPX2, targeting protein for Xklp2; Bax, Bcl‑2 associated X; Cyt‑C, cytochrome c; Apaf‑1, apoptotic protease activating factor 1.

Article Snippet: Mouse anti‐human Caspase‐9 antibodies (cat. no. 9508S; 1:1,000) were purchased from Cell Signaling Technology, Inc., rabbit anti‐human Bcl‐2 (cat. no. BA0412; 1:1,000) and cyclin B1 (CCNB1; cat. no. BA0766; 1:1,000) antibodies were purchased from Wuhan Boster Biological Technology, Ltd., rabbit anti‐human polo like kinase (PLK)‐1 antibodies (cat. no. 10305‐1‐AP; 1:1,000) and targeting protein for Xklp2 (TPX2) antibodies (cat. no. 11741‐1‐AP; 1:2,000) were purchased from Proteintech Group, Inc., and the rabbit anti‐human PLK‐4 antibodies (cat. no. A9863; 1:1,000) were purchased from ABclonal Biotech Co., Ltd.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: AURKA destruction is decoupled from its activity at mitotic exit but suppresses interphase activity

doi: 10.1101/850917

Figure Lengend Snippet: A pT288 antibody detects active AURKA only in mitotic cells. Cells were synchronized as described in Materials and Methods and blotted for pT288, total AURKA and other mitotic markers. B pT288 signal is sensitive to AURKA-specific inhibitor MLN8237 by IF on mitotic cells from a MeOH-fixed unsynchronized population (upper panel) or by immunoblot of STLC-arrested mitotic cells treated for 3 hours at the indicated doses (lower panel). AURKA-specific pT288 signal is restricted to centrosomes and spindle pole bodies (marked by γ-tubulin, TUBG1). Bars, 10 μm. See also Figure S1. C-E Quantification of pT288-AURKA during mitotic exit. C, D Unsynchronized cell populations were fixed and stained as in B . Cells were judged to be at different stages of mitosis according to DAPI staining ( C ) and scored for mean pT288 AURKA signal measured in a fixed ROI centred on TUBG1 signal at centrosomes or spindle poles ( D ). G2 and prophase (P), n=10; prometaphase (PM), n=15; metaphase (M), n=30; anaphase (A), n=30; and telophase (T), n=26. M vs A, not significant (n.s.); A vs T, p < 0.0001 (***), Students’ t-test. E Cells were synchronized in 5 μM STLC and released by checkpoint inhibition using 10 μM AZ3146, with extracts harvested at times indicated. These were examined by immunoblotting for AURKA, pT288-AURKA and TPX2 levels. Disappearance of Cyclin B1 (CCNB1) acts as marker for mitotic exit, level of vinculin (VCL) as loading control.

Article Snippet: Primary antibodies for immunoblot were as follows: AURKA mouse mAb (1:1000; Clone 4/IAK1, BD Transduction Laboratories), phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (1:1000; clone D13A11 XP® Rabbit mAb, Cell Signalling), rabbit polyclonal TPX2 antibody (1:1000; Novus Biological), Cdh1 mouse mAb (1:50; gift from T. Hunt and J. Gannon), CDC20 mouse mAb (1:1000; Santa Cruz sc13162), AURKB rabbit polyclonal antibody (1:1000; Abcam ab2254), mouse monoclonal Cyclin B1 (1:1000; BD 554177), DRP1 rabbit polyclonal (1:500; Bethyl lab), rabbit polyclonal Tubulin (1:2000; Abcam ab6046), mouse mAb anti-Vinculin (1:1000; clone hVIN-1, Sigma-Aldrich), rabbit anti-GFP (1:1000; 11814460001, Roche).

Techniques: Western Blot, Staining, Inhibition, Marker, Control

Journal: bioRxiv

Article Title: AURKA destruction is decoupled from its activity at mitotic exit but suppresses interphase activity

doi: 10.1101/850917

Figure Lengend Snippet: U2OS ( A,B ) and FZR1 KO ( C,D ) cells were transfected with TPX2(1-43)-CFP and synchronized through mitotic exit as described in the legend to . Quantitative immunoblotting of cell lysates shows that loss of pT288-AURKA during mitotic exit is delayed in the presence of TPX2(1-43) in both parental and FZR1 KO cells. Cyclin B1 (CCNB1) is used as marker for mitotic exit, level of vinculin (VCL) as loading control. Bar charts (B, D) show pT288 signal normalized against vinculin. Results presented are mean values from 3 independent experiments ± S.D. E AURKA inactivation is phosphatase dependent. U2OS cells undergoing mitotic exit were treated with PP1 inhibitor 3nM tautomycin 10 minutes after relief of checkpoint inhibition by AZ3146. Lysates harvested at the indicated time points after AZ3146 treatment were subject to immunoblot analysis.

Article Snippet: Primary antibodies for immunoblot were as follows: AURKA mouse mAb (1:1000; Clone 4/IAK1, BD Transduction Laboratories), phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (1:1000; clone D13A11 XP® Rabbit mAb, Cell Signalling), rabbit polyclonal TPX2 antibody (1:1000; Novus Biological), Cdh1 mouse mAb (1:50; gift from T. Hunt and J. Gannon), CDC20 mouse mAb (1:1000; Santa Cruz sc13162), AURKB rabbit polyclonal antibody (1:1000; Abcam ab2254), mouse monoclonal Cyclin B1 (1:1000; BD 554177), DRP1 rabbit polyclonal (1:500; Bethyl lab), rabbit polyclonal Tubulin (1:2000; Abcam ab6046), mouse mAb anti-Vinculin (1:1000; clone hVIN-1, Sigma-Aldrich), rabbit anti-GFP (1:1000; 11814460001, Roche).

Techniques: Transfection, Western Blot, Marker, Control, Inhibition

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: Identification cell cycle-related DEGs. ( A ) An intersection analysis of the TGCA-BRCA prognostic molecules and cell cycle-related DEGs. ( B – D ) The survival analysis of TPX2/UBE2C/CCNE2 with the indictor, overall survival (OS), disease specific survival (DSS), progress free interval (PFI), respectively. P <0.05 was considered significant.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques:

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: ( A ) The expression level of these three candidate cell cycle-related DEGs in BC or four BC subtypes (Basal-like; HER2; Luminal-A; Luminal-B) and normal patients. ( B ) Correlations between TPX2/UBE2C/CCNE2 expression and tumor stage in BRCA patients. P<0.05 is considered as difference. ( C , D ) Immunohistochemistry detecting the protein level of TPX2 in various stages. *P < 0.05. Scar bar: 100 μm.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: Expression profile and survival situation of TPX2 in BRCA. ( A – C ) Differential expression levels of TPX2 in BRCA from TCGA and GEO database. ( D ) The mRNA expression of TPX2 in breast cancer cell lines. ( E ) The differential protein level of TPX2 in breast tumor and normal tissues. **P < 0.01, ***P < 0.001.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: Univariate and multivariate Cox analyses of the correlation of TPX2 expression with overall survival (OS) among breast cancer patients.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: Correlation between TPX2 expression and the clinicopathological features of breast cancer patients for ( A ) T stage, ( B ) N stage, ( C ) M stage, ( D ) pathologic stage, ( E ) PAM50, ( F ) Age, ( G ) radiation therapy, ( H ) race and ( I ) histologic type. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: The relationship between TPX2 expression and tumor-infiltrating immune cells. ( A ) Stacked bar chart shows distribution of 22 immune cells in each sample. ( B ) Violin plot displays the differentially infiltrated immune cells between TPX2-High group and TPX2-Low group. Red color represents TPX2-High group, and blue color represents TPX2-Low group. ( C ) Correlation matrix of immune cell proportions. The red color represents positive correlation and the blue color represents negative correlation.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: ( A ) Correlation analysis between TPX2 expression and representative immune checkpoints from TCGA database. ( B ) The relationship between TPX2 expression and immune microenvironment. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: The relationship between TPX2 and PD-L1 in BRCA. ( A ) TIMER database illustrating the relationship between TPX2 and CD274 (PD-L1) in four BC subtypes. ( B ) RT-QPCR experiment illustrating the gene relationship between TPX2 and PD-L1. ( C ) Molecular docking simulating the amino acid interaction between TPX2 and PD-L1. ( D ) Flow cytometry illustrating the protein relationship between TXP2 and PD-L1. ( E ) Immunohistochemistry detecting the protein level of TPX2 and PD-L1 from tissue microarray. Scar bar: 100 μm. ***P < 0.001.

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Quantitative RT-PCR, Flow Cytometry, Immunohistochemistry, Microarray

Journal: Aging (Albany NY)

Article Title: Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer

doi: 10.18632/aging.205752

Figure Lengend Snippet: TPX2-related gene network, protein–protein interactions and enrichment analysis. ( A ) Top 5 TPX2-correlated genes in TCGA database, KIF4A, BUB1, PLK1, KIF20A and CKAP2L, Pearson’s correlation coefficients. ( B ) The heatmap revealing the correlation between TPX2 expression and these top 5 genes in various tumors. ( C ) PPI network construction of 48 experimentally verified TPX2-interacted proteins. ( D ) An intersection analysis of the TPX2-interacted and TPX2-correlated genes. ( E ) Enrichment analysis illustration based on the TPX2-interacted and TPX2-correlated genes. (BP: biological process; CC: cellular component; MF: molecular function; KEGG: Kyoto Encyclopedia of Genes and Genomes).

Article Snippet: Anti-TPX2 (ABclonal, A18327, China) performed at a concentration of 1:250, and the primary antibody against PD-L1 (Abcam, ab205921, USA) was diluted 1:200 in the process of immunohistochemistry assays.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: TPX2 is a direct target of miR-1294 in glioma cell lines. A. Predicted miR-1294 target sequence in the 3’-UTR of TPX2 mRNA. B. miR-1294 down-regulated the luciferase activity of the wild-type TPX2 3’UTR expression vector but did not reduce the expression of mutant TPX2. C. Expression of TPX2 in the CGGA public database. D. Expression of TPX2 in NBTs (n=5) and glioma specimens divided into LGG (n=8) and HGG (n=8). E. Expression of TPX2 in normal human astrocytes (NHAs) and four glioma cell lines (U87, U251, LN229, A172). F. Expression of TPX2 in U87 and U251 cells transfected with NC, miR-1294 mimic or miR-1294 inhibitor. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Sequencing, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Mutagenesis, Transfection

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: Down-regulation of TPX2 suppresses glioma cell proliferation, migration, and invasion and enhances chemosensitivity to temozolomide in vitro. A. qRT-PCR analysis of TPX2 expression in U87 and U251 cells transfected with NC or si-TPX2. B, C. The CCK-8 assay for U87 and U251 cells transfected with NC or si-TPX2. D. The migration assay for U87 and U251 cells transfected with NC or si-TPX2. E. The Transwell for U87 and U251 cells transfected with NC or si-TPX2. F, G. Cell viability was examined for U87 and U251 cells transfected with NC or si-TPX2 following TMZ treatments at various doses. H, I. Cell viability was examined every 24 hours in U87 and U251 cells transfected with NC or si-TPX2 following TMZ treatments at the indicated concentrations. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Migration, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: TPX2 overexpression reverses the suppressive effects of miR-1294 on glioma cells in vitro. A. qRT-PCR analysis of TPX2 expression in U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. B, C. The CCK-8 assay for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. D. The migration assay for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. E. The Transwell assay for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. F, G. Cell viability was examined for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2 following TMZ treatments at various doses. H, I. Cell viability was examined every 24 hours for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2 following TMZ treatments at the indicated concentrations. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Migration, Transwell Assay

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: miR-1294 inhibits glioma growth in vivo. A. Tumor formation was assessed in nude mice, and the tumors of U87 xenografts were excised. B. The volume of excised tumors according to the formula: V (mm3)=0.5 * a* b2 (a represents the longest axis and b the shortest axis). C. The weight of excised tumors. D, E. Expression of TPX2 in tumor tissue. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: In Vivo, Expressing