18g recombinant human gst ran protein sinobiological  (Sino Biological)

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: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Phosphorylation of Pyk2 kinase target proteins was analyzed in PSD extracts. GluN2B: * P = 0.0016 from s and * P = 0.0001 from c; Src: * P < 0.0001 from s and * P = 0.0003 from c; Tau: * P < 0.0001 from s and c; one-way ANOVA with Bonferroni test; n = 5 animals/group. b Pyk2 tyrosine phosphorylation was assessed on Y402 (* P = 0.0184 from s; ** P = 0.0040 from s and ** P < 0.0001 from 30 min rep; one-way ANOVA with Bonferroni test; n = 4–6 animals/group). c Overall Pyk2 tyrosine phosphorylation was assessed after Pyk2 pull down with a phospho-tyrosine-specific antibody (* P = 0.0089 from s; ** P = 0.0005 from s; one-way ANOVA with Bonferroni test; n = 4-6 animals/group). d Phosphorylation of immobilized recombinant his-GST-Src by Pyk2 derived from sham and MCAO lysates untreated or treated with recombinant USP2 was determined. * P = 0.0152 from s; # P = 0.0242 from i/untreated; one-way ANOVA with Bonferroni test; n = 4 animals/group. c contralateral, i ipsilateral, S serine, s sham, Y tyrosine. Data are expressed as mean ± s.e.m.

Article Snippet: For Src substrate phosphorylation, recombinant mouse his-GST-Src was first immobilized on glutathione sepharose 4B beads (GE Healthcare).

Techniques: Recombinant, Derivative Assay

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Phosphorylation of Pyk2 kinase target proteins was analyzed in PSD extracts. GluN2B: * P = 0.0016 from s and * P = 0.0001 from c; Src: * P < 0.0001 from s and * P = 0.0003 from c; Tau: * P < 0.0001 from s and c; one-way ANOVA with Bonferroni test; n = 5 animals/group. b Pyk2 tyrosine phosphorylation was assessed on Y402 (* P = 0.0184 from s; ** P = 0.0040 from s and ** P < 0.0001 from 30 min rep; one-way ANOVA with Bonferroni test; n = 4–6 animals/group). c Overall Pyk2 tyrosine phosphorylation was assessed after Pyk2 pull down with a phospho-tyrosine-specific antibody (* P = 0.0089 from s; ** P = 0.0005 from s; one-way ANOVA with Bonferroni test; n = 4-6 animals/group). d Phosphorylation of immobilized recombinant his-GST-Src by Pyk2 derived from sham and MCAO lysates untreated or treated with recombinant USP2 was determined. * P = 0.0152 from s; # P = 0.0242 from i/untreated; one-way ANOVA with Bonferroni test; n = 4 animals/group. c contralateral, i ipsilateral, S serine, s sham, Y tyrosine. Data are expressed as mean ± s.e.m.

Article Snippet: Pyk2 activity was assessed through its autophosphorylation level and capacity to phosphorylate recombinant mouse his-GST-Src (Sino Biological Inc., Chesterbrook, PA).

Techniques: Recombinant, Derivative Assay