Journal: Cells

Article Title: Functional Roles of Src Kinase Activity in Oocyte Maturation and Artificial Egg Activation in Xenopus laevis

doi: 10.3390/cells15030305

Figure Lengend Snippet: Time-dependent expression of recombinant xSrc proteins in X. laevis oocytes following mRNA microinjection, and progesterone-induced MAPK activation, CDK1/Cdc2 dephosphorylation, and GVBD occur normally in oocytes expressing recombinant xSrc proteins. ( A ) Left panels: Recombinant xSrc proteins, as indicated by asterisks (*), were detected by immunoprecipitation (IP) using mAb327 followed by immunoblotting (IB). All constructs became detectable approximately 4–5 h after mRNA injection. Middle panels: Total Src protein levels were examined using an antibody that recognizes both endogenous (**) and recombinant xSrc (*). Recombinant xSrc proteins were expressed at levels comparable to or higher than endogenous Src. Right panels: Representative bright-field images of oocytes at 0 h and 5 h after mRNA injection. No changes in surface pigmentation or germinal vesicle morphology were observed, indicating that forced expression of xSrc constructs did not induce premature maturation or morphological abnormalities. ( B ) Left panels: Immunoblotting for phosphorylated MAP kinase (pMAPK), as indicated by asterisks (*), showing time-dependent activation following exposure to 2 µM progesterone. Middle panels: Immunoblotting for CDK1/Cdc2 phosphorylated on the inhibitory tyrosine residue (pTyr-Cdc2), as indicated by asterisks (*). Dephosphorylation indicates activation of CDK1 and progression toward GVBD. Right panels: Representative images of oocytes at 0 h and 8 h after progesterone treatment. GVBD, visualized as disappearance of the germinal vesicle (arrowheads), occurred in all groups—including xSrcWT-, xSrcKA-, and xSrcKN-expressing oocytes—with kinetics similar to those of control oocytes. Together, these results indicate that forced expression of recombinant xSrc proteins, regardless of kinase activity, does not interfere with the hormonal maturation program of immature oocytes.

Article Snippet: Polyclonal antibodies against MAP kinase (MAPK) and phospho-MAPK were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Recombinant, Microinjection, Activation Assay, De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Construct, Injection, Residue, Control, Activity Assay

Journal: Cells

Article Title: Functional Roles of Src Kinase Activity in Oocyte Maturation and Artificial Egg Activation in Xenopus laevis

doi: 10.3390/cells15030305

Figure Lengend Snippet: Acceleration of progesterone-induced GVBD and increased tyrosine phosphorylation in oocytes expressing constitutively active xSrc. ( A ) Time course of GVBD in control oocytes and those expressing xSrcWT, xSrcKA, or xSrcKN. Oocytes expressing the constitutively active mutant xSrcKA displayed a modest but reproducible acceleration in GVBD onset compared with other groups. The percentage of GVBD was assessed at 1 h intervals following progesterone treatment. ( B ) Anti-phosphotyrosine immunoblot showing progesterone-induced tyrosine phosphorylation of multiple proteins. Oocytes expressing xSrcKA exhibited increased phosphorylation of several proteins relative to controls and other Src constructs. A strongly phosphorylated ∼50 kDa protein (**), distinct from recombinant xSrc itself (∼60 kDa; *) and MAPK (∼42 kDa; ***), was specifically enhanced in xSrcKA-expressing oocytes. This suggests the presence of an unidentified substrate whose phosphorylation is stimulated by elevated Src kinase activity.

Article Snippet: Polyclonal antibodies against MAP kinase (MAPK) and phospho-MAPK were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Expressing, Control, Mutagenesis, Western Blot, Construct, Recombinant, Activity Assay

Journal: Cells

Article Title: Functional Roles of Src Kinase Activity in Oocyte Maturation and Artificial Egg Activation in Xenopus laevis

doi: 10.3390/cells15030305

Figure Lengend Snippet: Differential requirements for Src kinase activity during egg activation induced by A23187, H 2 O 2 , or Cathepsin B. Matured oocytes (mRNA injection for 5 h + progesterone treatment for 8 h) were subjected to the indicated activation stimuli. ( A ) A23187-induced activation. Left: MAPK dephosphorylation assessed by anti-pMAPK immunoblotting. Right: Percentage of oocytes undergoing cortical contraction. All three xSrc-expressing groups activated efficiently, similar to controls, indicating that Src kinase activity is not required for Ca 2 +-ionophore-induced activation. ( B ) H 2 O 2 -induced activation. Left: MAPK dephosphorylation profiles. Right: Quantification of cortical contraction. xSrcKN-expressing oocytes showed markedly reduced activation compared to control, xSrcWT, and xSrcKA oocytes. ( C ) Cathepsin B-induced activation. Left: MAPK dephosphorylation following Cathepsin B treatment. Right: Activation rates based on cortical contraction. As with H 2 O 2 , xSrcKN-expressing oocytes displayed a strong reduction in responsiveness, demonstrating the requirement of Src kinase activity in membrane-associated activation signaling. In all panels, asterisks (*) indicate the positions of pMAPK.

Article Snippet: Polyclonal antibodies against MAP kinase (MAPK) and phospho-MAPK were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Activation Assay, Injection, De-Phosphorylation Assay, Western Blot, Expressing, Control, Membrane

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential microglial dynamics and neuroinflammation underlying neuropathic pain in the central nervous system: comparative insights from spinal cord injury and compressive myelopathy models

doi: 10.3389/fncel.2026.1769004

Figure Lengend Snippet: Expression of pain-related molecules in the spinal cord. (A) In the SCI model, p-p38 and p-ERK1/2 expression colocalized with CD11b at the lesion site peaked at 2 weeks post-injury and increased in the lumbar enlargement during the chronic phase (12 weeks). Scale bars = 50 μm. (B) In the DCM model, expression of p-p38 and p-ERK1/2 colocalized with CD11b progressively increased at the compression site, with little change in the lumbar enlargement. Scale bars = 50 μm.

Article Snippet: The primary antibodies (Abs) used were rabbit anti-phospho-p38 MAP kinase (p-p38) polyclonal Ab (1:400, Cell Signaling Technology, Beverly, MA); rabbit anti- p44/42 MAPK (p-ERK1/2) polyclonal Ab (Cell Signaling Technology); and rabbit anti-CD11b Ab (1:50, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential microglial dynamics and neuroinflammation underlying neuropathic pain in the central nervous system: comparative insights from spinal cord injury and compressive myelopathy models

doi: 10.3389/fncel.2026.1769004

Figure Lengend Snippet: Expression of pain-related molecules in the brain. (A) In the SCI model, there was significant expression of p-p38 and p-ERK1/2 colocalized with CD11b in the hippocampus and amygdala during the chronic phase (12 weeks) and in the thalamus at both acute and chronic time points. Scale bars = 50 μm. (B) In the DCM model, expression of pain-related molecules increased in the hippocampus, amygdala, and thalamus under moderate compression (18 weeks). Scale bars = 50 μm.

Article Snippet: The primary antibodies (Abs) used were rabbit anti-phospho-p38 MAP kinase (p-p38) polyclonal Ab (1:400, Cell Signaling Technology, Beverly, MA); rabbit anti- p44/42 MAPK (p-ERK1/2) polyclonal Ab (Cell Signaling Technology); and rabbit anti-CD11b Ab (1:50, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Effect of introducing point mutations on the biological activity of FGF1. a Serum-starved NIH 3T3 cells were treated with 10 ng/mL FGF1 variants for 15 min in the presence of heparin (10 U/mL). Activation of the downstream cascade was detected by immunoblotting using the following antibodies: anti-phospho-FRS2 (pFRS2) and anti-phospho-ERK1/2 (pERK1/2). Anti-ERK1/2 and anti-vinculin antibodies were used to confirm equal loading. Representative results are shown (n ≥ 3). The vertical lines in the last WB panel show the deleted wells. The original membranes, together with the method of trimming, are presented in Fig. S2. Densitometric analysis of pERK/ERK is presented in Fig. S3. b Effect of 20-h FGF1 variants stimulation (20 ng/mL) in the presence of 10 U/mL heparin on glucose uptake by 3T3-L1 adipocytes. Data are presented as mean ± SEM, n = 4. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001

Article Snippet: The following primary antibodies were used: anti-phospho-FGFR (Tyr653/Tyr654) (pFGFR) (#06–1433) from Millipore, anti-tubulin (#T6557) from Sigma-Aldrich, anti-FGFR1 (FGFR1) (#9740), anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase (pERK1/2) (#9101), anti-p44/42 MAP kinase (ERK1/2) (#9102), anti-phospho-FRS2α (Tyr196) (pFRS2) (#3864), anti-vinculin (#13901), anti-phospho-PLCγ1 (Tyr783) (pPLCγ) (#14,008), anti-PLCγ1 (#5690), anti-Glut1 (#73,015) and anti-FGF1 (#3139) from Cell Signaling Technology.

Techniques: Activity Assay, Activation Assay, Western Blot

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Impaired activation of signaling pathways by FGF1 variants due to reduced affinity for the FGFR1 (IIIc) receptor. a Serum-starved NIH 3T3 cells were stimulated with 10 ng/mL FGF1 variants in the presence of heparin (10 U/mL) for 15 min, and activation of downstream signaling cascades was detected by immunoblotting using the following antibodies: anti-phospho-FGFR (pFGFR), anti-phospho-PLCγ (pPLCγ), anti-phosphoFRS2 (pFRS2), anti-phospho-ERK1/2 (pERK1/2). Anti-ERK1/2, anti-FGFR1, anti-PLCγ and anti-γTubulin antibodies were used to confirm equal loading. Representative results are shown. Densitometric analysis is presented as mean ± SEM, n = 3/4. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001. b BLI analysis of the affinity of FGF1 variants for FGFR1-Fc (IIIc isoform). FGFR1-Fc was immobilized on a Protein A sensor and its interactions (association and dissociation) with selected FGF1 mutants were analyzed in the concentration range of 100–800 nM. Curves obtained by global fitting are marked in red. Representative results are shown (n ≥ 3). The equilibrium dissociation constant (K D ) was calculated from the saturation binding curve

Article Snippet: The following primary antibodies were used: anti-phospho-FGFR (Tyr653/Tyr654) (pFGFR) (#06–1433) from Millipore, anti-tubulin (#T6557) from Sigma-Aldrich, anti-FGFR1 (FGFR1) (#9740), anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase (pERK1/2) (#9101), anti-p44/42 MAP kinase (ERK1/2) (#9102), anti-phospho-FRS2α (Tyr196) (pFRS2) (#3864), anti-vinculin (#13901), anti-phospho-PLCγ1 (Tyr783) (pPLCγ) (#14,008), anti-PLCγ1 (#5690), anti-Glut1 (#73,015) and anti-FGF1 (#3139) from Cell Signaling Technology.

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Concentration Assay, Binding Assay