p re ss in vitro kinase assay src  (Sino Biological)

Journal: Nature Communications

Article Title: A hotspot phosphorylation site on SHP2 drives oncoprotein activation and drug resistance

doi: 10.1038/s41467-026-70060-8

Figure Lengend Snippet: a Kinase Library prediction heatmap based on the peptide primary sequence of the SHP2 Y62 phosphosite plotted on human kinome evolutionary tree. TK—Tyrosine kinases, CAMK—calcium calmodulin-dependent kinases, TKL—Tyrosine kinase-like, STE- sterile kinases, CK1—Casein Kinase 1. b SFK multi-kinase and selective inhibitors with targets shown, tested here. c , d Immunoblot analysis of ( c ) U-2 OS cells and ( d ) HCC827 cells treated with dasatinib and SU6656 at indicated concentrations, and DMSO. e , f Immunoblot analysis of HCC827 cells treated with ( e ) saracatinib, CH6953755, and PP2, and ( f ) double and triple combinations at indicated concentrations, and DMSO. g Immunoblot analysis of SYF knock out and wildtype MEFs. h In vitro kinase assays with recombinant SRC, YES1, and FYN kinases and recombinant SHP2 WT and SHP2 Y62F . The SHP2 pY542 antibody demonstrated nonspecific binding in the control conditions. All immunoblots are representative of one of the three independent experiments. The unit of molecular weight markers in Western blots is kDa. Source data are provided as source data file.

Article Snippet: SRC (Cat# S19-18G), YES1 (Cat# Y01-10G), and FYN (Cat# F15-10G) kinase proteins were purchased from SinoBiological.

Techniques: Sequencing, Phospho-proteomics, Sterility, Western Blot, Knock-Out, In Vitro, Recombinant, Binding Assay, Control, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src directly phosphorylates TAB1-Y481. A , the domain structure of TAB1 and the amino acid sequence around Y481. Conserved phosphorylation serine/threonine and tyrosine sites in the C-terminal regions are highlighted in green / blue and red . B , COS-7 cells were transfected with EGFP-tagged full-length (FL), N-terminal (N), or C-terminal (C) TAB1 and Src. C , COS-7 cells were transfected with EGFP-TAB1 and Src. Dasatinib (0.1 μM) was added 1 h before cells were harvested. D , COS-7 cells were transfected with TAB1-C or its Y481F mutant (YF) and Src. Cell lysates and immunoprecipitates with an anti-GFP antibody were immunoblotted with anti-phosphotyrosine and anti-GFP antibodies. The expression of active Src was detected by an anti-phospho-Src family kinase (Y419) antibody ( B – D ). E , an in vitro kinase assay was performed using recombinant GST-TAB1-C and active GST-Src proteins. F , COS-7 cells were transfected with EGFP-TAB1 with SFKs, including Src, Fyn, Lyn, and Lck. G , HCT-116-Src cells were treated with 10 ng/ml doxycycline (Dox) for 24 h. TAB1-Y481 phosphorylation and other proteins were detected by an anti-phospho-Y481-TAB1 antibody and the antibodies described above, respectively ( E – G ). H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD from three independent experiments. p values were calculated using Welch’s two-tailed t test. ∗ p < 0.05.

Article Snippet: The recombinant human GST-TAB1-C protein (WT and Y481F) derived from Escherichia coli ( ) was reacted with the recombinant human active GST-Src kinase derived from insect cells (Carna Bioscience) at 30 °C for 30 min in 30 μl of reaction buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl 2 , 0.2 mM ATP, 2 mM DTT, 20 mM β-glycerophosphate, and 0.1 mM sodium orthovanadate.

Techniques: Sequencing, Phospho-proteomics, Transfection, Mutagenesis, Expressing, In Vitro, Kinase Assay, Recombinant, Quantitative Proteomics, Two Tailed Test

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 activates the Wnt/β-catenin signaling pathway in mouse hippocampal neuronal HT22 cells. ( A ) The SuperTOPFlash reporter gene was transfected into HT22 cells, and the cells were treated with solvent control (DMSO) and gradient concentrations of ISX9 (2.5-40 μM) for 24 h. Subsequently, the cells were lysed, and the fluorescence intensity was detected and normalized based on the value of the β-gal. ( B ) HT22 cells were treated with control and gradient concentrations of ISX9 (2.5-40 μM) for 24 h. The expression levels of endogenous β-catenin and activated β-catenin proteins were detected by Western blot. Quantitative analysis was performed by ImageJ and normalized relative to GAPDH values, and results were presented on the right. ( C ) HT22 cells were treated with control and 20 μM ISX9 for 24 h, respectively, followed by precipitation of cell lysates with an anti-AXIN1 antibody. The interaction of AXIN1 with LRP6 or β-catenin was detected by immunoblotting. ( D ) HT22 cells were treated with control or gradient concentrations of ISX9 (2.5-20 μM) for 24 h, respectively. After cell lysis, total RNA was extracted, and cDNA was obtained by reverse transcription. RT-qPCR was performed to detect the mRNA expression levels of Wnt target genes NOTUM, AXIN2 and SURVIVIN. The data shown are the mean ± SD (n = 3). Statistical significance was tested by one-way ANOVA followed by the Tukey test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated that there was a significant difference compared with the control group

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Transfection, Solvent, Control, Fluorescence, Expressing, Western Blot, Lysis, Reverse Transcription, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 enhances the proliferation, and promotes the expression of stemness-related Wnt target genes in hippocampal neuronal cells. HT22 cells were treated with control or gradient concentrations of ISX9 for 24 h, respectively. The cell viability ( A ) and proliferation ( B ) of HT22 cells were detected by MTT and BrdU assays. ( C ) HT22 cells were treated with control or ISX9 (2.5-20 μM) for 24 h, respectively. The cells were lysed to extract total RNA, which was then subjected to reverse transcription for cDNA synthesis. RT-qPCR was performed to detect the mRNA expression levels of stemness-related Wnt target genes LGR5 and SOX2. ( D - F ) HT22 cells were treated with control and 20 μM of ISX9 for 24 h. Then the cells were fixed and followed by IF staining with DAPI and anti-β-catenin antibody ( D ) or anti-LGR5 antibody ( E ) or anti-SOX2 antibody ( F ). Alexa Fluor 594 conjugated goat anti-mouse IgG antibodies were used as secondary antibodies. Scale bar, 20 μm. The results of the quantitative fluorescence analysis were presented on the right. The data shown are the mean ± SD (n = 3). Statistical significance was tested by one-way ANOVA followed by the Tukey test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated that there was a significant difference compared with the control group

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Expressing, Control, Reverse Transcription, cDNA Synthesis, Quantitative RT-PCR, Staining, Fluorescence

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 activates Wnt/β-catenin signaling in the hippocampus of AD mice. ( A ) Expression levels of total β-catenin and activated β-catenin protein in hippocampal tissues of WT, WT/ISX9, AD and AD/ISX9 group mice. The below bars show the results of quantification of the immunoblotting images by ImageJ and normalization relative to GAPDH values. ( B ) Protein was extracted from the hippocampal tissues of mice, followed by precipitation with an anti-AXIN1 antibody. The interaction of AXIN1 with LRP6 or β-catenin was detected by immunoblotting. ( C ) Total RNA extracted from mouse hippocampal tissues of mice was reverse transcribed to obtain cDNA, and then RT-qPCR was performed to detect the mRNA expression levels of Wnt target genes NOTUM, AXIN2 and SURVIVIN

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR