Journal: Oncology reports

Article Title: MEK1 and MEK2 isoforms regulate distinct functions in pancreatic cancer cells.

doi: 10.3892/or_00000853

Figure Lengend Snippet: Figure 1. MEK1 and MEK2 mRNA expression in PC-1.0 cells after shRNA- mediated knockdown. Expression of MEK1 shRNAs in PC-1.0 cells significantly decreased MEK1 mRNA levels compared with the empty vector but showed no inhibition of MEK2 expression. Similarly, MEK2 shRNAs markedly decreased MEK2 mRNA levels but did not affect MEK1 expression. ß-actin served as the normalisation control.

Article Snippet: Rabbit polyclonal antibodies raised against epitopes of human MEK1, MEK2, and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.

Techniques: Expressing, shRNA, Knockdown, Plasmid Preparation, Inhibition, Control

Journal: Oncology reports

Article Title: MEK1 and MEK2 isoforms regulate distinct functions in pancreatic cancer cells.

doi: 10.3892/or_00000853

Figure Lengend Snippet: Figure 2. MEK1 and MEK2 protein levels in PC-1.0 cells after MEK1 or MEK2 knockdown. MEK1 shRNA expression in PC-1.0 cells significantly decreased MEK1 protein levels compared with the empty vector but had no effect on MEK2 expression. MEK2 shRNA expression markedly decreased MEK2 protein levels but had no effect on MEK1 protein. ß-actin was detected as a loading control.

Article Snippet: Rabbit polyclonal antibodies raised against epitopes of human MEK1, MEK2, and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.

Techniques: Knockdown, shRNA, Expressing, Plasmid Preparation, Control

Journal: Oncology reports

Article Title: MEK1 and MEK2 isoforms regulate distinct functions in pancreatic cancer cells.

doi: 10.3892/or_00000853

Figure Lengend Snippet: Figure 3. Cell morphology of MEK1-knockdown and MEK2-knockdown PC-1.0 cells. MEK2 knockdown apparently induced cell aggregation in PC-1.0 cells (C) compared with the control cells (A). In contrast, MEK1 knockdown showed no significant effect on cell growth patterns in PC-1.0 cells (B). The fluorescent images show stable transfection of empty control vector (D), MEK1-shRNA (E), and MEK2-shRNA (F).

Article Snippet: Rabbit polyclonal antibodies raised against epitopes of human MEK1, MEK2, and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.

Techniques: Knockdown, Control, Stable Transfection, Plasmid Preparation, shRNA

Journal: Oncology reports

Article Title: MEK1 and MEK2 isoforms regulate distinct functions in pancreatic cancer cells.

doi: 10.3892/or_00000853

Figure Lengend Snippet: Figure 4. Cell proliferation in MEK1- and MEK2-knockdown PC-1.0 cells. Knockdown of MEK1 exerted an apparent anti-proliferative effect on PC- 1.0 cells after 48 and 72 h. On the contrary, MEK2 knockdown exhibited no significant inhibition of cell growth or proliferation in PC-1.0 cells.

Article Snippet: Rabbit polyclonal antibodies raised against epitopes of human MEK1, MEK2, and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.

Techniques: Knockdown, Inhibition

Journal: Oncology reports

Article Title: MEK1 and MEK2 isoforms regulate distinct functions in pancreatic cancer cells.

doi: 10.3892/or_00000853

Figure Lengend Snippet: Figure 5. Effects of MEK1 and MEK2 knockdown on mitotic arrest in PC- 1.0 cells. MEK1 knockdown decreased the percentage of cells in S phase, but no change to the cell cycle was observed in MEK2-knockdown PC-1.0 cells. Black bar, control PC-1.0 cells with empty vector; dotted bar, MEK1- knockdown PC-1.0 cells; white bar, MEK2-knockdown PC-1.0 cells. S, significant; NS, not significant.

Article Snippet: Rabbit polyclonal antibodies raised against epitopes of human MEK1, MEK2, and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.

Techniques: Knockdown, Control, Plasmid Preparation

Journal: Oncology reports

Article Title: MEK1 and MEK2 isoforms regulate distinct functions in pancreatic cancer cells.

doi: 10.3892/or_00000853

Figure Lengend Snippet: Figure 6. Invasive capability in MEK1- and MEK2-knockdown PC-1.0 cells. Knockdown of MEK2 significantly inhibited the invasive capability of PC-1.0 cells compared with cells expressing the empty vector. However, MEK1 knockdown did not affect invasive capability. S, significant; NS, not significant.

Article Snippet: Rabbit polyclonal antibodies raised against epitopes of human MEK1, MEK2, and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies.

Techniques: Knockdown, Expressing, Plasmid Preparation

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 1. A schematic drawing illustrates the four signaling pathways utilized by TGFb to affect cellular function. This diagram was prepared based on reviews [18, 19, 21]. Original articles that describe the discovery of the key molecules depicted in these pathways can be found in these reviews. MEKKs, MAP/ERK kinase kinases, which include MEKK1, MEKK2, MEKK3, and others; JNK, c-Jun NH2-terminal kinase also known as Jun kinase or stress-activated protein kinase, SAPK; JNKK, c-Jun NH2-terminal kinase kinase; MKK3, MAP kinase kinase 3; MEK1/2, MAP/ERK kinase 1 and MAP/ERK kinase 2; ERK1/2, extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Protein-Protein interactions, Cell Function Assay

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 4. Relative expression of the TGFb- upstream signal transducers in Sertoli and germ cells isolated from 20-day-old rat testes. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli and germ cells. Im- munoblots were performed in parallel ex- periments to assess the relative protein levels of these signal transducers in Sertoli and germ cells using the corresponding specific antibodies (see Materials and Methods). (C). B) This figure shows the corresponding densitometrically scanned results using autoradiograms or immuno- blots such as those shown in A and C. Re- sults are expressed as mean 6 SD using three batches of cells from three different experiments normalized against S16. Each experiment had triplicate cultures. These analyses revealed that results of immuno- blots are consistent with the RT-PCR data. Statistical analysis was performed by Stu- dent t-test comparing germ cells with the corresponding Sertoli cells, which was ar- bitrarily set at one. *, Significantly different from Sertoli cells by Student t-test, P , 0.05; **, significantly different from Sertoli cells by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 5. Developmental regulation of the steady-state mRNA levels of the TGFb up- stream signal transducers in Sertoli (A, B) and germ (C, D) cells. RT-PCR was per- formed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli cells (A) and germ cells (C) during maturation. B, D) The cor- responding densitometrically scanned re- sults using autoradiograms such as those shown in A and C. Results are expressed as mean 6 SD using two batches of cells normalized against S16 from two different experiments. Each experiment had tripli- cate cultures. ns, Not significantly different from cultures isolated from rats at 20 days of age in B and 5 or 10 days of age in D, which was arbitrarily set at one, by Stu- dent t-test; *, significantly different by Stu- dent t-test, P , 0.05; **, significantly dif- ferent by Student t-test, P , 0.01; nd, not detectable.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 6. Changes in the steady-state mRNA levels of TGFb upstream signal transducers in the testis during develop- ment. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in testes during maturation. B) This panel shows the corresponding densi- tometrically scanned results using autora- diograms such as those shown in A. Re- sults are expressed as mean 6 SD using testes from three different rats normalized against S16. ns, Not significantly different from rats at 5 days of age, which was arbi- trarily set at one, by Student t-test; *, signif- icantly different by Student t-test, P , 0.05; **, significantly different by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 8. Change in the steady-state mRNA and protein levels of the TGFb upstream signal transducers when the Sertoli cell TJ barrier was assembled in vitro in the ab- sence (control) and presence (test) of TGFb3. Sertoli cells (0.5 3 106 cells/cm2) cultured on Matrigel-coated dishes in the absence (A, C, E) or presence (B, D, F) of TGFb3 (3 ng/ml) were terminated by RNA STAT-60 or lysed in SDS sample buffer at specified time points. Cell lysates (;200 mg protein) from each time point were re- solved by SDS-PAGE under reducing con- ditions using 10% T SDS-polyacrylamide gels. Immunoblotting was performed to as- sess changes in the levels of Smad2, Cdc42, Rac2, N-Ras, and MEKK2 in the absence (A, E) and presence (B, F) of re- combinant TGFb3 (3 ng/ml). RT-PCR was performed to assess changes in the MEKK2 steady-state mRNA level in the absence (C) or presence (D) of recombinant TGFb3 (3 ng/ml). G, H) Corresponding densito- metrically scanned results using autoradio- grams and immunoblots shown in C–F. Results are expressed as mean 6 SD from three separate experiments using different batches of cells and normalized against S16. Each time point had duplicate cul- tures. ns, Not significantly different by AN- OVA, in which each sample at a given time point was compared with samples of all other time points within the same ex- perimental group; *, significantly different by ANOVA, P , 0.01; D, days.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Control, Cell Culture, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Recombinant

Journal: Signal transduction and targeted therapy

Article Title: IGF-1R inhibition induces MEK phosphorylation to promote survival in colon carcinomas.

doi: 10.1038/s41392-020-0204-0

Figure Lengend Snippet: Fig. 5 Inactivation of MEK1 or MEK2 stimulates AKT phosphorylation. a, b Knockdown of MEK1 or MEK2 boosts AKT phosphorylation. Cells were transfected with scrambled siRNA, mek1 siRNA, or mek2 siRNA. After 48 h, cell lysates were subjected to western blot analysis. a Representative images are shown. b Densitometric quantification of phospho-AKT levels in (a). The ratio of phospho-AKT/GAPDH of transfection with scrambled siRNA is designated as 1. Data from three independent experiments were analyzed by one-sample t-test (mean ± SD; *P < 0.05). c, d Inhibition of MEK1/2 activation induces AKT phosphorylation. HCT116 cells were treated with 0, 1, 5, and 10 μM of U0126 for 72 h. The cell lysates were used to examine the levels of phospho-AKT. c Representative western blot images are shown. b Densitometric quantification of phospho-AKT and phospho-p70S6K1 levels in (c). The ratio of phospho-AKT/GAPDH or phospho-p70S6K1 in cells with 0 μM of U0126 is designated as 1. Data from three independent experiments were analyzed by one-sample t-test (mean ± SD; *P < 0.05; **P < 0.01)

Article Snippet: The MEK2 (67410–1-Ig) antibody was purchased from Proteintech (1:4,000 dilution).

Techniques: Phospho-proteomics, Knockdown, Transfection, Western Blot, Inhibition, Activation Assay

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