Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 1. TRIM28 expression is elevated in cervical cancer tissues and cell lines. (A) Analysis from the Oncomine database showing that TRIM28 mRNA expression is significantly elevated in cervical cancer tissues (n=40) compared with that in normal tissues (n=5). The data were obtained from a previous study (35). (B) TRIM28 mRNA levels in 20 paired cervical cancer samples and adjacent normal tissues examined by qRT‑PCR. (C) Immunohistochemical staining of normal and cervical cancer tissues with anti-TRIM28 antibody. The expression levels of TRIM28 in human cervical cancer cells and normal human cervical tissues were measured by qRT‑PCR (D) and western blotting (E). Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 3. TRIM28 enhances cervical cancer cell tumorigenicity. (A) Tumor volumes were measured on the indicated days. (B) At the end of the experiment, the weights were measured. (C) The proliferation index (right) was determined based on the percentage of Ki67-positive cells (left). (D) Western blot analysis of TRIM28 and p-mTOR protein expression in indicated tumor xenografts. Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Western Blot, Expressing

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 2. TRIM28 promotes cervical cancer cell proliferation by accelerating the cell cycle. (A) Western blot analysis of TRIM28 protein expression in TRIM28-infected cells and TRIM28 shRNA-infected cells. GAPDH was used as the loading control. (B) Effects of TRIM28 upregulation or downregulation on the proliferation of SiHa or CaSki cells analyzed by the MTT assay. (C) Representative micrographs (left) and quantification (right) of colonies formed by the indicated cervical cancer cell lines. (D) Cell cycle progression of the indicated cervical cancer cell lines analyzed by flow cytometry. (E) Representative images (left) and quantification (right) of BrdU-positive cells in the indicated cervical cancer cell lines. Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Western Blot, Expressing, Infection, shRNA, Control, MTT Assay, Flow Cytometry

Journal: Oncology reports

Article Title: TRIM28 promotes cervical cancer growth through the mTOR signaling pathway.

doi: 10.3892/or.2018.6235

Figure Lengend Snippet: Figure 4. TRIM28 promotes cervical cancer cell proliferation via the mTOR signaling pathway. (A) Western blot analysis of TRIM28, p-mTOR(S2448), mTOR, p-S6K1(T389), and S6K1 in TRIM28 shRNA-infected CaSki cells and TRIM28-infected SiHa cells in the presence or absence of 20 nM evero- limus (EVE) for 48 h. GAPDH served as the loading control. (B-D) Cell proliferation, cell cycle, and BrdU incorporation were analyzed in TRIM28-infected SiHa cells in the presence or absence of EVE for 48 h. Data are presented as means ± SD. *P<0.05.

Article Snippet: After blocking the endogenous peroxidase with 3% hydrogen peroxide, the sections were washed with Tris-buffered saline (TBS) and then incubated with a primary antibody targeting TRIM28 (Proteintech, Hubei, China) for 1 h at 37 ̊C.

Techniques: Western Blot, shRNA, Infection, Control, BrdU Incorporation Assay

Journal: Molecular Cell

Article Title: Antibody RING-Mediated Destruction of Endogenous Proteins

doi: 10.1016/j.molcel.2020.04.032

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti TRIM28 , Cell Signaling Technology , 4124.

Techniques: Recombinant, DNA Ligation, Staining, Ubiquitin Proteomics, Transfection, Gel Extraction, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Mass Spectrometry, Phospho-proteomics, Software, Targeted Proteomics

Journal: Frontiers in Immunology

Article Title: Phosphorylation of TRIM28 Enhances the Expression of IFN-β and Proinflammatory Cytokines During HPAIV Infection of Human Lung Epithelial Cells

doi: 10.3389/fimmu.2018.02229

Figure Lengend Snippet: Inhibition of p38 and MSK1 ablates TRIM28 S473 phosphorylation. A549 cells were pre-treated with inhibitors for (A) p38 (SB202190), (B) MEK (U0126), (C) MSK1 (SB747651), and (D) MK2 (PF-3644022) at 0.1 μM, 0.5 μM, 1 μM, 5 μM and 10 μM before infection with SC35M at an MOI of 20 for 10 h and further incubation with the inhibitors. Lysates were examined with a phospho-specific antibody for TRIM28 S473 phosphorylation. Infection was verified by detection of the viral PB1 protein. Tubulin and total TRIM28 served as loading controls. MEK and MK2 inhibition was controlled by detection of ERK T202/Y204, and HSP27 S82 phosphorylation, respectively. (E) A549 cells were pre-treated with p38 (SB202190), MEK (U0126), MSK1 (SB747651) and MK2 (PF-3644022) inhibitors at 1 μM for 30 min. Pre-treated cells were transfected with 500 ng viral or cellular in the presence of the corresponding inhibitors. Lysates were harvested 4 h p.t. and TRIM28 S473 phosphorylation was analyzed by western blot.

Article Snippet: Proteins were transferred to nitrocellulose membranes and detected by using primary antibodies targeting tubulin (Sigma, Germany), PB1 (GeneTex, USA), TRIM28, TRIM28 S473-P, TRIM28 S824-P (Abcam, UK), CREB S133-P, RIG-I, eIF2α S51-P, ERK1/2, ERK1/2 T202/Y204-P, HSP27, HSP27 S82-P (Cell signaling Technologies, USA) and anti-mouse or anti-rabbit IgG secondary antibodies either conjugated to fluorophores (Licor, Germany) or horseradish peroxidase (Cell Signaling Technology, USA).

Techniques: Inhibition, Phospho-proteomics, Infection, Incubation, Transfection, Western Blot

Journal: Frontiers in Immunology

Article Title: Phosphorylation of TRIM28 Enhances the Expression of IFN-β and Proinflammatory Cytokines During HPAIV Infection of Human Lung Epithelial Cells

doi: 10.3389/fimmu.2018.02229

Figure Lengend Snippet: Inhibition of p38 and MSK1 ablates TRIM28 S473 phosphorylation. A549 cells were pre-treated with inhibitors for (A) p38 (SB202190), (B) MEK (U0126), (C) MSK1 (SB747651), and (D) MK2 (PF-3644022) at 0.1 μM, 0.5 μM, 1 μM, 5 μM and 10 μM before infection with SC35M at an MOI of 20 for 10 h and further incubation with the inhibitors. Lysates were examined with a phospho-specific antibody for TRIM28 S473 phosphorylation. Infection was verified by detection of the viral PB1 protein. Tubulin and total TRIM28 served as loading controls. MEK and MK2 inhibition was controlled by detection of ERK T202/Y204, and HSP27 S82 phosphorylation, respectively. (E) A549 cells were pre-treated with p38 (SB202190), MEK (U0126), MSK1 (SB747651) and MK2 (PF-3644022) inhibitors at 1 μM for 30 min. Pre-treated cells were transfected with 500 ng viral or cellular in the presence of the corresponding inhibitors. Lysates were harvested 4 h p.t. and TRIM28 S473 phosphorylation was analyzed by western blot.

Article Snippet: Proteins were transferred to nitrocellulose membranes and detected by using primary antibodies targeting tubulin (Sigma, Germany), PB1 (GeneTex, USA), TRIM28, TRIM28 S473-P, TRIM28 S824-P (Abcam, UK), CREB S133-P, RIG-I, eIF2α S51-P, ERK1/2, ERK1/2 T202/Y204-P, HSP27, HSP27 S82-P (Cell signaling Technologies, USA) and anti-mouse or anti-rabbit IgG secondary antibodies either conjugated to fluorophores (Licor, Germany) or horseradish peroxidase (Cell Signaling Technology, USA).

Techniques: Inhibition, Phospho-proteomics, Infection, Incubation, Transfection, Western Blot