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: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A binds to Ras and activates the Ras/ERK signaling to mediate RSK activation (A) The volcano plot analysis results for the FAM65A high-expression and low-expression groups from the TCGA database were shown. (B) The KEGG and GO results were shown. (C) The GSEA results were shown. (D) GSEA on DEGs between the FAM65A high-expression group and low-expression group in the Reactome database were shown. (E) IP was performed to detect the binding of FAM65A and Ras/p-RSK. (F) IP was performed to detect the binding of Ras and FAM65A/p-RSK. (G) Immunofluorescence was performed to detect the co-localization of FAM65A and Ras. Scale bars, 20 μm. (H) Western blot analysis the Ras and p -ERK expression in FAM65A knockdown or overexpression cells. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Expressing, Binding Assay, Immunofluorescence, Western Blot, Knockdown, Over Expression

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Ras/ERK signaling activation was indispensable for FAM65A-mediated RSK activation and CRC progression (A) Western blot analysis of Ras and p -ERK expression in HCT116-FAM65A cells treated with 10 μM Abd-7, or without treatment. (B) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of Abd-7, n = 3, ∗∗∗ p < 0.001. (C) Colony formation assay performed on HCT116-FAM65A cells treated with Abd-7 or not. (D) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (E) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 100 μm. (F) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (G) Western blot analysis of Ki-67, cleaved Caspase 3, Bcl-2, and Bax expression in HCT116-FAM65A cells treated with Abd-7 or not. (H) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (I) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (J) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 50 μm. (K) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (L) Results from the wound healing assay performed on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (M) Quantitative analysis of the wound healing assay results, n = 3, ∗∗∗ p < 0.001. (N) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with Abd-7 or not. (O) Proposed model of FAM65A in CRC progression. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Knockdown of FAM65A inhibits tumor progression in vivo (A) LOVO-shCtrl and LOVO-shFAM65A cells were administered into the fourth fat pad of nude mice, and the resulting tumor growth curves were subsequently generated, n = 5, ∗ p < 0.05. (B) The tumors excised from mice across various experimental groups are presented. (C) Hematoxylin and Eosin (HE) staining results of lung tissue from the different groups is displayed. (D) A quantitative analysis of metastatic lung nodules is provided, n = 5, ∗∗ p < 0.01. (E) IHC results for FAM65A, Ki-67, p -RSK, p -ERK, Ras, N-cadherin, vimentin, cleaved Caspase 3, ZO-1, and E-cadherin in tumor tissues are illustrated. (F) A quantitative analysis of the IHC results is included. Data are presented as mean ± SEM of biologically independent experiments, n = 5, ∗∗∗ p < 0.001.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Knockdown, In Vivo, Generated, Staining

Journal: PLoS ONE

Article Title: Potent Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

doi: 10.1371/journal.pone.0043612

Figure Lengend Snippet: Top: Foreground fluoresence intensities of secondary goat anti-rabbit IgG (H+L) conjugated with DyLight680 antibody to each overlapping 13-mer peptide in the microarray are shown (means and SD). Bottom: Raw fluorescence images of representative peptide microarrays are shown. Each array is framed by a fusion tag (Flag) peptide (DYKDDDDKGG, 72 red spots) and influenza virus hemagglutinin (HA) epitope tag peptide (YPYDVPDYAG, 72 green spots) which were used as internal controls.

Article Snippet: After pre-swelling the arrays for 10 min in standard buffer (phosphate-buffered saline [PBS], pH 7.4+0.05% Tween 20) and 60 min in Rockland blocking buffer (Rockland Immunochemicals, Inc., Gilbertsville, PA), the peptide microarrays were initially incubated with the secondary goat anti-rabbit IgG (H+L) conjugated with DyLight680 antibody at a dilution of 1∶5000 for 60 min at room temperature to verify that no significant background interactions occurred with the peptide arrays.

Techniques: Microarray, Fluorescence

Journal:

Article Title: Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38

doi: 10.1093/emboj/cdf361

Figure Lengend Snippet: Fig. 5. The RalGDS–Ral pathway mediates insulin- and EGF-induced activation of ATF2-dependent gene expression. (A) The effects of insulin and MMS on Ral activity. A14 cells were transiently transfected with 3 µg of pMT2-HA-Ral. At 24 h after transfection, the cells were serum-starved overnight followed by stimulation with either 10 nM insulin (15 min) or 1 mM MMS (2 h). Total cell extracts (750 µg of protein) were incubated with 15 µg of GST–RalBD pre-coupled to glutathione beads to recover GTP-bound Ral. Beads were washed extensively, and collected Ral was detected by immunoblotting with HA antibody. (B) Insulin- and EGF-induced activation of ATF2-dependent transcription is inhibited by RasN17 and RalN28. A14 cells were transiently transfected with 2 µg of either the cJun–ATF2-dependent luciferase reporter 5×jun2-tata or the tata-luciferase control, in the presence or absence of 2 µg of expression vectors for RasN17 and RalN28, or an empty control vector. At 20 h after transfection, the cells were stimulated for 6 h with 10 nM insulin or 1 mM MMS. Depicted is the relative luciferase activity (RLU) ± SD. (C) Dominant-negative Ral inhibits insulin-induced p38 phosphorylation. A14 cells were transiently transfected with 0.5 µg of pMT2-HA-p38 in the presence or absence of 1.5 µg of pMT2-HA-RalN28, or an empty expression vector as described in Figure 2A. Subsequently, the cells were serum-starved and treated with either 10 nM insulin or 500 mM NaCl (O.S.). Total cell extracts were prepared after 15 min, and analyzed by SDS–PAGE/immunoblotting. For better comparison, a relatively short exposure of osmotic shock-induced HA-phospho-p38 is shown. (D) Activation of Ral by RlfCAAX induces p38 phosphorylation. A14 cells were transfected with 0.5 µg pMT2-HA-p38 in the presence or absence of 0.125 µg of HA-RlfCAAX, or an empty vector (–) as described above. At 24 h after transfection, the cells were serum-starved and, after an additional 24 h, total cell lysates were prepared and analyzed by SDS–PAGE and immunoblotting. (E) Activation of Ral by RlfCAAX induces p38 and JNK kinase activity. A14 cells were transfected with 0.5 µg of expression vectors encoding HA-tagged p38, JNK or ERK, respectively, in the presence or absence of 0.125 µg of HA-RlfCAAX expression vector, or an empty vector (–) as described above. At 24 h after transfection, the cells were serum-starved and, after an additional 24 h, total cell lysates were prepared. Lysates were immunoprecipitated with an HA antibody, after which HA-associated ATF2 Thr71 kinase activity was measured using GST–ATF2 as substrate (see Materials and methods). (F) Activation of Ral by RlfCAAX induces ATF2 Thr69 + 71 phosphorylation. A14 and JNK–/– cells were left untreated (–) or transfected with 0.5 µg of pMT2-HA-ATF2 in the presence or absence of 0.125 µg of RlfCAAX expression vector. Fugene reagent was used in order to obtain high levels of transfection efficiency (>40%). At 24 h after transfection, cells were serum-starved overnight, and incubated for a further 24 h in the presence or absence of 10 µM U0126 prior to preparation of cell lysates and analysis by SDS–PAGE and immunoblotting. Note that HA-ATF2 and HA-RlfCAAX (detected by the HA antibody) have nearly the same molecular weight. (G) RlfCAAX enhances transactivation by ATF2 via ATF2 Thr69 and Thr71. A14 cells were transiently transfected with 2 µg of 5×GAL4-E4-luciferase reporter plasmid together with 2 µg of pRSV-GAL4-ATF2 expression vectors containing full-length (wt) ATF2, or the corresponding domain in which Thr69 (T69A), Thr71 (T71A) or both (T69/71A) are replaced by alanine. In addition to these GAL4 fusion constructs, 3 µg of pMT2-RlfCAAX, or an empty expression vector was co-transfected. At 40 h after transfection, cells were harvested and analyzed for luciferase activity. The fold activation depicted represents the ratio between luciferase activity in the presence and absence of RlfCAAX. Values represent the mean ± SD.

Article Snippet: The other antibodies used were: phospho-specific ATF2 Thr69 + 71, ATF2 Thr71, p38 Thr180/Tyr182, ERK hr202/Tyr204 (Cell Signaling Technology), p38 (N-20), ATF-2 (C-19), cJun (H-79), ERK (K-23), donkey anti-goat IgG–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology), monoclonal anti-HA antibody 16B12 (BabCO), anti-GST (Amersham Pharmacia Biotech), phospho-specific JNK Thr183/Tyr185, goat anti-rabbit and goat anti-mouse IgG–HRP conjugate (Promega).

Techniques: Activation Assay, Expressing, Activity Assay, Transfection, Incubation, Western Blot, Luciferase, Plasmid Preparation, Dominant Negative Mutation, SDS Page, Immunoprecipitation, Molecular Weight, Construct

Journal:

Article Title: Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38

doi: 10.1093/emboj/cdf361

Figure Lengend Snippet: Fig. 4. Insulin- and EGF-induced ATF2 Thr71 mono-phosphorylation is mediated by ERK. (A) The main insulin-induced ATF2 N-terminal kinase activity co-purifies with ERK1/2 after MonoQ anion-exchange chromatography. Total cell lysates from A14 cells treated for 15 min with either 10 nM insulin or 500 mM NaCl (O.S.) were separated on a MonoQ column using a linear gradient of NaCl (dotted line). Fractions were analyzed for in vitro ATF2 kinase activity (filled circles) as described in Materials and methods, and for the presence of JNK, ERK1/2 and p38 by SDS–PAGE and immunoblotting. (B) Insulin induces ERK-associated ATF2 N-terminal kinase activity. Serum-starved A14 cells were either untreated or treated for 15 min with 10 nM insulin, with or without 15 min pre-treatment with 20 µM PD98059, as indicated. Total cell lysates were immunoprecipitated with antibodies that recognize both ERK1 and ERK2, and subsequently assayed for ATF2-kinase activity. (C) Insulin-induced ERK and p38 activities differ in their ATF2 Thr69 and Thr71 kinase activities. Partially purified ERK and p38 preparations from insulin-stimulated A14 cell extracts (MonoQ fractions 12 and 17, respectively) were analyzed for in vitro kinase activity, using either wild-type (wt) or mutant GST–ATF2 fusion proteins in which Thr69 (T69A), Thr71 (T71A) or Thr69 + 71 (T69/71A) were replaced by alanine. The phosphorylation state subsequently was monitored by SDS–PAGE followed by autoradiography and immunoblotting using phospho-specific antibodies followed by enhanced chemiluminescence (ECL). (D) Quantification of 32P incorporation into GST–ATF2 by MonoQ fractions 12 and 17 in in vitro kinase assays as described in (C). The relative activity (mean ± SD) shown represents the 32P incorporation in the various mutant GST–ATF2 substrates relative to that in the wild-type GST–ATF2 protein (set at 100% for both fractions 12 and 17). (E) Recombinant active ERK only phosphorylates ATF2 Thr71 efficiently. Recombinant ERK (10 U; Calbiochem) was compared with MonoQ fraction 13 and total cell lysate from insulin-treated A14 cells for its ATF2 kinase potential using GST–ATF2 as a substrate. The phosphorylation state of GST–ATF2 Thr69 + 71 and GST–ATF2 Thr71 subsequently was monitored by SDS–PAGE/immunoblotting using phospho-specific antibodies. Equal loading of the GST–ATF2 substrate was verified by reprobing the filters with GST antibodies.

Article Snippet: The other antibodies used were: phospho-specific ATF2 Thr69 + 71, ATF2 Thr71, p38 Thr180/Tyr182, ERK hr202/Tyr204 (Cell Signaling Technology), p38 (N-20), ATF-2 (C-19), cJun (H-79), ERK (K-23), donkey anti-goat IgG–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology), monoclonal anti-HA antibody 16B12 (BabCO), anti-GST (Amersham Pharmacia Biotech), phospho-specific JNK Thr183/Tyr185, goat anti-rabbit and goat anti-mouse IgG–HRP conjugate (Promega).

Techniques: Activity Assay, Chromatography, In Vitro, SDS Page, Western Blot, Immunoprecipitation, Purification, Mutagenesis, Autoradiography, Recombinant