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: 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:

Article Title: MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

doi:

Figure Lengend Snippet: Dependence on IKKγ and expression of MAP3Ks and GCK in wild-type (+/+) and mutant (−/−) ES cells. (A) Wild-type and Ikkγ- (only a single Ikkγ allele was disrupted, but as Ikkγ is an x-linked gene and the ES cells we used were XO, this resulted in complete loss of IKKγ expression) ES cells were left untreated (0 time point) or treated with TNFα or IL-1 as described above. At the indicated times, cell lysates were prepared and IKK activity was determined by using an IKKα monoclonal antibody and immunocomplex kinase assay. (B) Exponentially growing ES cells or 3T3 fibroblasts were lysed, and equal amounts (50 μg) of total cell lysates were resolved by SDS/PAGE, transferred to a nitrocellulose membrane, and probed with antibodies to ASK1, TAK1, MEKK2, or GCK (Santa Cruz Biotechnology). All of these antibodies were found to specifically recognize their cognate antigens based on immunoblot analysis of cells transiently transfected with expression vectors for the different kinases (not shown).

Article Snippet: At the indicated times, cell lysates were prepared and IKK activity was determined by using an IKKα monoclonal antibody and immunocomplex kinase assay. ( B ) Exponentially growing ES cells or 3T3 fibroblasts were lysed, and equal amounts (50 μg) of total cell lysates were resolved by SDS/PAGE, transferred to a nitrocellulose membrane, and probed with antibodies to ASK1, TAK1, MEKK2, or GCK (Santa Cruz Biotechnology).

Techniques: Expressing, Mutagenesis, Activity Assay, Kinase Assay, SDS Page, Western Blot, Transfection