Journal: Cell reports

Article Title: THBS2-producing matrix CAFs promote colorectal cancer progression and link to poor prognosis via the CD47-MAPK axis.

doi: 10.1016/j.celrep.2025.115555

Figure Lengend Snippet: Figure 7. THBS2 induces EMT and tumor progression through activation of the MAPK/ERK5 pathway (A) Volcano plot displaying differentially expressed genes (DEGs) between CAF-THBS2 and CAF-NC. (B) Gene ontology (GO) enrichment analysis of DEGs highlights significant enrichment in ECM remodeling pathways. (C) Ridge plot showing the enriched pathway results from proteomics analysis. (D) Heatmap of phosphoproteomic data involved in the MAPK pathway. (E) Correlation analysis showing a positive association between THBS2 expression and MAPK7 expression (top) and phosphorylation (bottom) in CRC samples from the CPTAC-COAD dataset. (F) Bar plot depicting correlations between THBS2 expression and genes associated with cell proliferation and EMT in the CPTAC-COAD and TCGA-CRC datasets.

Article Snippet: The membranes were then incubated overnight at 4 C with the following primary antibodies: a-SMA (1:50000, Cat No. 80008-1-RR, Proteintech), FAP (1:1000, Cat No. ab207178, Abcam), CREB3L1 (1:1000, Cat No. 67671-1-Ig, Proteintech), THBS1 (1:1000, Cat No. 18304-1-AP, Proteintech), THBS2 (1:1000, Cat No. PA5-80123, Invitrogen), COMP (1:5000, Cat No. ab231977, Abcam), ZEB1 (1:1000, Cat No. 21544-1-AP, Proteintech), E-cadherin (1:20000, Cat No. 20874-1-AP, Proteintech), N-cadherin (1:3000, Cat No. 22018-1-AP, Proteintech), Vimentin (1:5000, Cat No. 10366-1-AP, Proteintech), Phospho-Erk5 (1:2000, Cat No. AF8146, Affinity), Erk5 (1:1000, Cat No. 3372S, Cell Signaling Technology), MEK5 (1:1000, Cat No. 91670S, Cell Signaling Technology), Cyclin D1 (1:10000, Cat No. 26939-1-AP, Proteintech), CD47 (1:2000, Cat No. 20305-1-AP, Proteintech) and Beta Actin (1:50000, Cat No. 66009-1-Ig, Proteintech).

Techniques: Activation Assay, Expressing, Phospho-proteomics

Journal: Central-European Journal of Immunology

Article Title: Down-regulation of SHP2 promotes neutrophil autophagy and inhibits neutrophil extracellular trap formation to alleviate asthma through the ERK5 pathway

doi: 10.5114/ceji.2024.143691

Figure Lengend Snippet: Primer sequences used in the study

Article Snippet: After transfer, the membrane was rinsed once in 1×PBST, and then the membrane was completely immersed in the blocking solution and shaken for 90 min. Primary antibodies were diluted in a ratio with 1×PBST: beclin1 (BECN1, AWA00914, 1 : 1000, Abiowell), LC3II/I (AWA01019, 1 : 1000, Abiowell), p62 (66184-1-Ig, 1 : 10000, Proteintech), SHP2 (ab32083, 1 : 1000, Abcam), MPO (22225-1-AP, 1 : 1000, Proteintech), neutrophil elastase (ELANE, AWA42997, 1 : 1000, Abiowell), peptidyl arginine deiminase 4 (PADI4, 17373-1-AP, 1 : 1000, Proteintech), ERK5 (AWA00874, 1 : 1000, Abiowell), and β-actin (AWA80002, 1 : 5000, Abiowell).

Techniques:

Journal: Central-European Journal of Immunology

Article Title: Down-regulation of SHP2 promotes neutrophil autophagy and inhibits neutrophil extracellular trap formation to alleviate asthma through the ERK5 pathway

doi: 10.5114/ceji.2024.143691

Figure Lengend Snippet: Inhibition of NET formation in neutrophil-like cells through down-regulating the SHP2/ERK5 pathway. A ) RT-qPCR and WB were performed to detect SHP2 expression. * p < 0.05 vs. si-NC and # p < 0.05 vs. si-SHP2 + oe-NC B ) IF staining was employed to detect cit-H3 expression. C, D ) Levels of dsDNA and MPO activity in neutrophil-like cells were measured. * p < 0.05 vs. si-NC and # p < 0.05 vs. si-SHP2 + oe-NC E, F ) Protein expression of MPO, ELANE, PADI4 and ERK5 was analyzed using WB. G ) Expression levels of SHP2 and ERK5 were analyzed using RT-qPCR and WB. * p < 0.05 vs. si-NC and # p < 0.05 vs. si-SHP2 + oe-NC H ) Cit-H3 expression was detected using IF. I, J ) Levels of dsDNA and MPO activity in neutrophils were measured. K ) WB was performed to analyze the protein expression of MPO, ELANE, and PADI4 in neutrophil-like cells. * p < 0.05 vs. si-NC and # p < 0.05 vs. si-SHP2 + oe-NC

Article Snippet: After transfer, the membrane was rinsed once in 1×PBST, and then the membrane was completely immersed in the blocking solution and shaken for 90 min. Primary antibodies were diluted in a ratio with 1×PBST: beclin1 (BECN1, AWA00914, 1 : 1000, Abiowell), LC3II/I (AWA01019, 1 : 1000, Abiowell), p62 (66184-1-Ig, 1 : 10000, Proteintech), SHP2 (ab32083, 1 : 1000, Abcam), MPO (22225-1-AP, 1 : 1000, Proteintech), neutrophil elastase (ELANE, AWA42997, 1 : 1000, Abiowell), peptidyl arginine deiminase 4 (PADI4, 17373-1-AP, 1 : 1000, Proteintech), ERK5 (AWA00874, 1 : 1000, Abiowell), and β-actin (AWA80002, 1 : 5000, Abiowell).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Staining, Activity Assay

Journal: Central-European Journal of Immunology

Article Title: Down-regulation of SHP2 promotes neutrophil autophagy and inhibits neutrophil extracellular trap formation to alleviate asthma through the ERK5 pathway

doi: 10.5114/ceji.2024.143691

Figure Lengend Snippet: Down-regulation of SHP2 promoted autophagy and inhibited formation of NETs in peripheral blood-derived neutrophils from asthma patients. A ) Expression levels of SHP2 and ERK5 were detected by RT-qPCR and WB in neutrophils. B ) IF and WB were utilized to detect the expression of LC3, BECN1 and p62 to assess neutrophil autophagy. * p < 0.05 vs. si-NC C ) IF and WB were utilized to detect the expression of LC3, BECN1 and p62 to assess neutrophil autophagy. D ) Expression of cit-H3 was detected using IF. * p < 0.05 vs. si-NC E, F ) Levels of dsDNA and MPO activity in neutrophils were measured by ELISA. G ) WB was performed to analyze the protein expression of MPO, ELANE, and PADI4 in neutrophils. * p < 0.05 vs. si-NC

Article Snippet: After transfer, the membrane was rinsed once in 1×PBST, and then the membrane was completely immersed in the blocking solution and shaken for 90 min. Primary antibodies were diluted in a ratio with 1×PBST: beclin1 (BECN1, AWA00914, 1 : 1000, Abiowell), LC3II/I (AWA01019, 1 : 1000, Abiowell), p62 (66184-1-Ig, 1 : 10000, Proteintech), SHP2 (ab32083, 1 : 1000, Abcam), MPO (22225-1-AP, 1 : 1000, Proteintech), neutrophil elastase (ELANE, AWA42997, 1 : 1000, Abiowell), peptidyl arginine deiminase 4 (PADI4, 17373-1-AP, 1 : 1000, Proteintech), ERK5 (AWA00874, 1 : 1000, Abiowell), and β-actin (AWA80002, 1 : 5000, Abiowell).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, IF-P, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression

doi: 10.3390/ijms25063273

Figure Lengend Snippet: Proteomic studies: immunoprecipitation. ( A ) Schematic representation of ERK5, showing the regions recognized by antibodies. ( B ) Whole cell extracts from NP9 cells were immunoprecipitated with the antibodies indicated in ( A ) in the absence or presence of an excess of the fusion protein or peptide against which the antibodies were generated. SDS-PAGE (5–10% gradient gel) was conducted with the samples, and protein bands were visualized by silver staining. Bands highlighted with red and green asterisks were cut out and analyzed using MALDI-TOF MS. ( C ) Data obtained by mass spectrometry (from the bands marked with red and green asterisk) were analyzed using the Mascot search engine against the Swiss-Prot database. Individual scores higher than 59 were considered significant ( p < 0.05). ( D , E ) Matching peptides in the GAC and KGA sequences are highlighted in red, and % coverage values are indicated.

Article Snippet: Cells lacking human ERK5 were obtained using a CRISPR/Cas9 knockout kit (reference KN200655) from OriGene (Rockville, MD, USA).

Techniques: Immunoprecipitation, Generated, SDS Page, Silver Staining, Mass Spectrometry

Journal: International Journal of Molecular Sciences

Article Title: ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression

doi: 10.3390/ijms25063273

Figure Lengend Snippet: Immunofluorescence and subcellular fractionation. ( A ) NP9 cells were incubated with 75 nM of MitoTracker (red) for 30 min at 37 °C in culture to mark the mitochondria. The cells were then fixed and stained with anti-C-terminus or anti-N-terminus ERK5 antibodies, followed by a Cy2-labeled secondary antibody (green). Dapi (blue) was used for staining nuclei. The images were captured with a confocal microscope. Scale bar, 25 µm. Experiments were replicated at least three times. ( B ) Subcellular fractionation was performed on NP9 cells. First, an amount of 80 µg of whole cell extracts (as a control) and 50 µL of either a cytoplasmic fraction (CF) or a mitochondrial fraction (MF) were resolved using SDS-PAGE and analyzed by Western blotting. ERK5 was detected by using the C-terminus antibody. KGA and GAC were detected using the anti-KGA/GAC rabbit antibody. COX IV and GAPDH were used as markers of mitochondrial and cytosolic fractions, respectively. ( C ) NP18, NP31, A549, and BT549 cells were labeled with MitoTracker (red) in a culture and then fixed and stained with the anti-ERK5 C-terminus antibody, followed by a Cy2-labeled secondary antibody (green). Cell nuclei were stained with Dapi (blue). Images were taken with a confocal microscope. Scale bar, 25 µm. Experiments were replicated at least three times.

Article Snippet: Cells lacking human ERK5 were obtained using a CRISPR/Cas9 knockout kit (reference KN200655) from OriGene (Rockville, MD, USA).

Techniques: Immunofluorescence, Fractionation, Incubation, Staining, Labeling, Microscopy, Control, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression

doi: 10.3390/ijms25063273

Figure Lengend Snippet: The co-immunoprecipitation of ERK5 and KGA/GAC and the colocalization of ERK5 and KGA/GAC in situ by immunofluorescence . ( A ) NP9 cell extracts were immunoprecipitated with the anti-ERK5 antibody, and immunocomplexes were assessed using SDS-PAGE and visualized by Western blotting with an anti-GAC antibody. In addition, 50 µg of whole cell extracts was also analyzed. ( B ) The same as in panel A, with the differences that the immunoprecipitation was performed with the anti-GAC antibody and Western blotting was carried out with the anti-ERK5 antibody. ( C , D ) Co-immunoprecipitation data between ERK5 and KGA, using the anti-KGA/GAC rabbit antibody. Additionally, a 50 µg amount of whole cell extracts was also analyzed. ( E ) Representative confocal microscopy images using anti-ERK5 C-terminus (rabbit) and anti-KGA/GAC (mouse) primary antibodies. Cy3-conjugated anti-rabbit (red) and Alexa Fluor ® 488-conjugated anti-mouse (green) antibodies were used as secondary antibodies. Nuclei were stained with Dapi (blue). Scale bar, 50 µm. ( F ) Representative confocal microscopy images using anti-ERK5 C-terminus (mouse) and anti-GAC (rabbit) primary antibodies. Alexa Fluor ® 568-conjugated anti-mouse (red) and Cy2-conjugated anti-rabbit (green) antibodies were used as secondary antibodies. Nuclei were visualized using Dapi (blue). Scale bar, 50 µm. Immunofluorescence experiments were replicated at least three times.

Article Snippet: Cells lacking human ERK5 were obtained using a CRISPR/Cas9 knockout kit (reference KN200655) from OriGene (Rockville, MD, USA).

Techniques: Immunoprecipitation, In Situ, Immunofluorescence, SDS Page, Western Blot, Confocal Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression

doi: 10.3390/ijms25063273

Figure Lengend Snippet: The effect of ERK5 depletion on GLS expression levels and its enzymatic activity . The downregulation of ERK5 in the ( A ) NP9 and ( B ) BT549 cell lines was achieved by lentiviral infection with pLKO.1 vectors carrying shRNA sequences for ERK5 (#62 and #75). A nonsense shRNA sequence (shC) was used as a control. An amount of 1 mg of cell extracts was immunoprecipitated with the Pro1 anti-ERK5 antibody, and ERK5 expression was detected by Western blotting with the C-terminus anti-ERK5 antibody. For the detection of KGA/GAC expression levels, 50 µg of NP9 and 100 µg of BT549 cell extracts were used, and Western blots were performed with anti-KGA/GAC rabbit antibody. GAPDH or CNX were used as loading controls. Glutaminase enzymatic activity was determined in ( C ) NP9 and ( D ) BT549 cells with downregulated ERK5. Data are given as the mean ± SD values of three independent experiments with three replicates each. pLKO-shKGA/GAC#37 cells were used as internal controls. ( E ) ERK5 knockout was performed via CRISPR/Cas9 in NP9 cells. SC stands for negative scramble control. ERK5 and KGA/GAC expression and ( F ) glutaminase enzymatic activity were detected in the same way as in the previous panels of this Figure. In all cases, enzyme activity was represented as the % of control (shC for shRNA and SC for the Crispr/Cas9 experiments). p -values are indicated. ( G ) Changes in mRNA KGA/GAC levels upon ERK5-KO in NP9 cells (clones #6 and #7) were determined via a qRT-PCR. Data were normalized to GAPDH expression and represented as relative quantity (RQ) values of mRNA expression with respect to a control (SC), which was assigned an RQ value of 1. RQ values were calculated according to the 2 −ΔΔCt method. Error bars for RQ values were calculated by the equations RQ min = 2 −(ΔΔCt+SD) and RQ max = 2 −(ΔΔCt−SD) . Experiments were repeated twice independently, with three replicates each. p -values are indicated. ns: not significant.

Article Snippet: Cells lacking human ERK5 were obtained using a CRISPR/Cas9 knockout kit (reference KN200655) from OriGene (Rockville, MD, USA).

Techniques: Expressing, Activity Assay, Infection, shRNA, Sequencing, Control, Immunoprecipitation, Western Blot, Knock-Out, CRISPR, Clone Assay, Quantitative RT-PCR