Journal: International Journal of Biological Sciences

Article Title: LncRNA SLCO4A1-AS1 predicts poor prognosis and promotes proliferation and metastasis via the EGFR/MAPK pathway in colorectal cancer

doi: 10.7150/ijbs.38041

Figure Lengend Snippet: Proposed schematic model illustrating the role of SLCO4A1-AS1 in regulating CRC by EGFR/MAPK signaling pathway. SLCO4A1-AS1 influences EGFR/MAPK signaling pathway by promoting the expression of EGFR, KRAS, BRAF, MEK, ERK, MAP3K1 and its corresponding phosphorylated protein levels, which further affect the proliferation, migration and invasion of CRC cells.

Article Snippet: The membrane was incubated with primary antibody overnight at 4 °C, including EGFR (1:2000, Abcam), P-EGFR (1:2000, Abcam), KRAS (1:2000, Abcam), BRAF (1:2000, Abcam), MEK1/2 (1:2000, Abcam), P-MEK1/2 (1:1000, Proteintech), ERK (1:2000, Abcam), P-ERK (1:2000, Abcam), MAP3K1 (1:700, Proteintech), P-MAP3K1 (1:1000, Proteintech), β-actin (1:700, Proteintech), incubate for 2 hours at room temperature with anti-rabbit secondary antibody.

Techniques: Expressing, Migration

Journal: Molecular cancer

Article Title: A novel polypeptide CAPG-171aa encoded by circCAPG plays a critical role in triple-negative breast cancer.

doi: 10.1186/s12943-023-01806-x

Figure Lengend Snippet: Fig. 6 CAPG-171aa interacts with STK38 activating the downstream MEK1/2-ERK1/2 pathway via MEKK2. (A) MDA-MB-231 and MDA-MB-468 cell lysates were IP with anti-MEKK2 antibody followed by detection with anti-MEKK2, STK38, and SMURF1 antibody. (B) MDA-MB-231 and MDA-MB-468 were trans fected with CAPG-171aa-FLAG. Whole-cell lysates were IP with anti-SMURF1 and IgG antibodies followed by detection with anti-FLAG, STK38, SMURF1, and GAPDH antibodies. (C-D) Before being treated with MG132, MDA-MB-231, and MDA-MB-468 were transfected with CAPG-171aa-FLAG and circCAPG KD plasmids. Ubiquitination and protein expression levels of MEKK2 were assayed in CAPG-171aa OE (C) and circCAPG KD (D) MDA-MB-231 and MDA- MB-468. (E) Ubiquitination and protein expression levels of MEKK2 were assayed in circCAPG KD MDA-MB-231 and MDA-MB-468 through pulse-chase experiments with cycloheximide. (F) IB of MEKK2, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in circCAPG KD MDA-MB-231 and MDA-MB-468. (G) IB of p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in OE STK38 MDA-MB-231 and MDA-MB-468. (H) IB of MEKK2, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in CAPG-171aa, OE-STK38 and OE-STK38/CAPG-171aa transfected MDA-MB-231 and MDA-MB-468. All data were representative of at least three biological replicates and shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: MDA-MB-231 and MDA-MB-468 cells were treated with 10 μg/mL MG132, respectively, (Solarbio, IM0310) for 12 h. Cell lysates were obtained using PierceTM IP lysis buffer (Thermo Fisher Scientific, USA) supplemented with a cocktail (Thermo Fisher Scientific, USA) and then incubated with anti-MEKK2 antibody (Proteintech, USA) and Protein A/G beads overnight at 4°C.

Techniques: Transfection, Ubiquitin Proteomics, Expressing, Pulse Chase

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 1. Ser-84-independent attenuation of PPAR activity upon TPA stimulation. HEK-293 cells were transiently cotransfected with mutants of PPAR and MEK1-GFP together with a 3 PPRE-lucif- erase reporter plasmid. Twenty-four hours after transfection, cells were starved for 16 h and then treated with rosiglitazone (Rosi, 10 M), TPA (100 nM), or 25 M PD098059 for another 24 h before harvesting. Luciferase activity was detected as described in Materials and

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Activity Assay, Plasmid Preparation, Transfection, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 2. Regulation of the PPAR target gene ACO by MEK1. (A) MKN45 cells were starved for 16 h and stimulated with TPA (100 nM), rosiglitazone (Rosi, 1 M), or a combination of both for 24 h. Total RNA of these cells was extracted, and quantitative RT-PCR was performed with ACO primers as indicated in Materials and Methods. Results are expressed as mean n-fold increase standard deviation of mRNA (normalized to S12 RNA) compared to vehicle control (n 3 independent experiments). (B) To study the effect of MEKs on rosiglitazone-induced expression of ACO, MKN45 cells were tran- siently transfected with empty vector, WT MEK1, DN MEK1, and NES-MEK1. Two days later, the cells were starved (0.1% FCS) for 16 h and were stimulated with either rosiglitazone (Rosi, 1 M) or DMSO as a control, both for 24 h. The quantitative RT-PCR was performed on total RNA as indicated in Materials and Methods. Results are expressed as mean n-fold increase of mRNA (normalized to S12) compared to vehicle control of cells transfected with empty vector of three samples.

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Quantitative RT-PCR, Standard Deviation, Control, Expressing, Transfection, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 4. The interaction between MEK1 and PPAR is increased upon TPA and rosiglitazone stimulation. (A) HEK-293 cells cotrans- fected with GFP-PPAR and MEK1-HA were serum starved (16 h) and then stimulated with 100 nM TPA or 10 M rosiglitazone for the indicated times. CoIP was carried out with HA Ab, and the amount of coimmunoprecipitated PPAR was determined by Western blotting (IB) with GFP Ab. The amounts of MEK1-HA and GFP-PPAR in the extracts were determined by Western blotting with the indicated Abs. (B) Densitometric analyses of CoIP experiments shown in panel A. Values represent n-fold increases standard deviations in complex formation compared to vehicle-treated unstimulated cells (n 3).

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 5. Determination of the site of MEK1 and PPAR interaction. (A and B) HEK-293 cells were transiently cotransfected with WT-PPAR, PPARC5, and PPAR459 together with MEK1-GFP. Cells were starved and stimulated and CoIP was performed as described for Fig. 3A. Precipitates and original extracts were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (IB) with the indicated Abs. Representative gels (A) and densitometric analyses (B) are shown. Values represent n-fold increases standard deviations in complex formation compared to vehicle-treated unstimulated cells (n 3). (C and D) The same experiments and analyses as described for panels A and B, except that the cells were cotransfected with WT PPAR together with WT MEK1-GFP, CA MEK1-GFP, and DN MEK1-GFP. (E and F) The same experiments and analyses as described for panels A and B, except that cells were cotransfected with WT PPAR together with DN MEK1-GFP (DN), CA MEK1-GFP (CA), NES-MEK1-GFP (NES), and 3-5A-MEK1-GFP (KKK). (G and H) HEK-293 cells were transiently cotransfected with WT PPAR together with MEK1-GFP, and IP was carried out as described in the presence of the indicated peptides (400 M each). The peptides used (see Materials and Methods) were derived form the CRS/CD of ERK (E-CRS), the CRS-like sequence of PPAR (P-CRS), D box of PPAR (P-DBox), leucine-rich region of ERK1 (E-LR), and leucine-rich region of PPAR (P-LR).

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Polyacrylamide Gel Electrophoresis, Western Blot, Derivative Assay, Sequencing

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 6. Cytosolic retention of PPAR upon overexpression of MEK1-GFP. Cells were grown on coverslips and transiently cotrans- fected with PPAR constructs and the indicated constructs of MEKs fused to GFP as described in Materials and Methods. Thirty-two hours after transfection, the cells were serum starved for 16 h, fixed, and stained for PPAR and DAPI, which were visualized using a fluores- cence microscope (Nikon, Japan) at 400 magnifications. (A) HeLa cells cotransfected with WT PPAR and WT MEK1-GFP in the indi- cated ratios. (B) COS7 cells cotransfected with PPAR together with

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Over Expression, Construct, Transfection, Staining, Microscopy

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 7. MEKs induce the nuclear export of PPAR upon TPA stimulation. (A) CHO cells were grown on coverslips as above. Following serum-starvation, CHO cells were treated with TPA (250 nM, 15 and 30 min), or with U0126 (5 M) 15 min prior to TPA (250 nM, 15 min). The cells were stained with PPAR Ab or PPAR Ab and DAPI, and the localization was assessed by fluorescence microscopy as above. (B) HeLa cells were cotransfected with GFP to identify transfected cells together with either combination of 4 siRNA oligonucleotides of MEK1 and 2 (Si-MEK) or with vector (pSuper) alone and then grown on coverslips as described above. Seventy-two hours after transfection, the cells were serum starved (0.1% FCS, 16 h) and then were treated with TPA (250 nM, 30 min) or left untreated. The cells were stained with polyclonal rabbit PPAR and DAPI and developed with rhodamine-conjugated anti-rabbit secondary Ab. The localization of PPAR was visualized by a fluorescence microscopy as above. (C) HeLa cells were transfected either with pSuper or with combination of 4 siRNA oligonucleotides of MEK1 and 2. Ninety hours after transfection, the cells were extracted, and the endogenous proteins were subjected to Western blot analysis using C-terminus MEK and actin Abs.

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Staining, Microscopy, Transfection, Plasmid Preparation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Interaction with MEK Causes Nuclear Export and Downregulation of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1128/mcb.00601-06

Figure Lengend Snippet: FIG. 9. PPAR nuclear export is mediated by MEKs. Subconfluent, serum-starved (0.1% FCS, 16 h) HeLa cells were treated as follows: (A) stimulated with TPA (250 nM, 15 and 30 min) or left untreated; (B) stimulated with TPA (250 nM, 15 and 30 min) or pretreated with U0126 (5 M) 15 min prior to addition of TPA (250 nM, 15 min) or vehicle control. (C) HeLa cells were transiently transfected with combination of 4 siRNA vectors of MEK1 and 2 (SiMEK) or with vector (pSuper) control. Four days after transfections, the cells were serum starved for 16 h and treated with TPA (250 nM, 15 min) () or left untreated (). (D) HeLa cells were preincubated for 45 min with interfering cell-permeable peptide CRSPerE (E-CRS) or the control permeable peptide LRPerP (P-LR) or left untreated, followed by TPA stimulation (250 nM, 15 min). (E) HeLa cells were treated with TPA (250 nM, 15 min) or with LMB (5 ng/ml, 60 min) plus TPA (250 nM, 15 min) or left untreated. All treated cells were extracted and subjected to cellular fractionation as described. The lysates were Western blotted with PPAR Ab and with tubulin Ab and histone H1 (histone) Ab as markers for the cytosolic (Cyt) and nuclear (Nuc) fractions, respectively. (F) The amount of PPAR in the cytosol was quantitated using densitometry, and the results are shown in a bar graph (n 3).

Article Snippet: Monoclonal PPAR (E-8, sc-7273), polyclonal PPAR (H-100, sc-7196), MEK1 (Sc6250), MEK1 C terminal (Sc 219) histone H1 and tubulin E-19 Abs were from Santa Cruz Biotechnology (CA), the hemagglutinin (HA) and green fluorescent protein (GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany), and pMEK1/2 Ab was from Cell Signaling (Beverly, MA).

Techniques: Control, Transfection, Plasmid Preparation, Cell Fractionation, Western Blot