Journal: BioMed Research International

Article Title: Shenmai Injection Supresses Glycolysis and Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant A549/DDP Cells via the AKT-mTOR-c-Myc Signaling Pathway

doi: 10.1155/2020/9243681

Figure Lengend Snippet: Antibodies used in the experiment.

Article Snippet: p-Akt (Ser473) , 33281 M , Bioss , Mouse , 1 : 500.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: Ten novel human JAK3 tyrosine auto-phosphorylation sites identified via mass spectrometry. ( a ) Schematic model of 10 tyrosine phosphorylation sites identified following the JAK3 autokinase assay and their location within JAK functional domains. ( b ) Representative mass spectra for JAK3-Y841 (*) are shown for novel tyrosine phosphorylation sites. ( c ) Sequence alignment showing five amino acids up-stream and down-stream of JAK3-Y841 (*) across multiple species. ( d ) Partial sequence alignment for human JAK1 (Y894), JAK2 (Y868), and Tyk2 (Y916) aligned against JAK3-Y841 (*). ( e ) Ribbon structure of JAK3 JH1 domain (PDB 5TTV) depicting ATP (purple) and Y841 (red) within the kinase domain N-lobe.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Phospho-proteomics, Mass Spectrometry, Functional Assay, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: JAK3 Y841 and the homologous JAK1 Y894 are critical for the activation of STAT5B. ( a ) Hek293 cells were transfected with 10 μg WT (lane a) or mutant JAK3 plasmids (lanes b–i) and incubated for 48 h at 37 °C. Cells were harvested after 48 h, immunoprecipitated for JAK3 and Western blotted for total pY. Shown is a representative blot from n = 2 independent experiments. Individual points for replicate experiments are provided in . ( b ) Hek293 cells were transfected with 2 µg of WT (lane a) or mutant JAK3 plasmids (lanes b–i) with 2 µg of STAT5B plasmids (lanes b–i) and harvested after 48 h at 37 °C. Western blot was performed using anti-pYSTAT5 and reblotted for total STAT5 to confirm the equal protein expression and loading of STAT5B. Shown is a representative blot from n = 3 independent experiments. ( c ) Densitometry analysis using LI-COR Image Studio Digits was performed. Anti-pYSTAT5 signal was normalized against total STAT5 protein and graphed as the fold change in relation to WT JAK3. Error bars represent the mean +/− standard deviation from three independent experiments (n = 3). A paired Student’s t -test was performed between the densitometry values of the activated STAT5 from WT JAK3 and the individual JAK3 mutants. The individual data points for n = 3 replicate experiments and statistical analysis are provided in . ( d ) Hek293 cells not transfected (NT, lane a) or transfected with c-myc-tagged JAK1 WT (lanes b,d), kinase dead (K907E, lane c) or Y894F (lane e), and WT STAT5B (lanes c–e) was immunoprecipitated and Western blotted, as indicated with anti-pY or anti-myc. Whole cell lysates (WCL) obtained prior to immunoprecipitation were subjected to Western blot with anti-pY STAT5 or anti-STAT5, as indicated.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Activation Assay, Transfection, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: Phosphospecific monoclonal anti-pY841 JAK3 selectively detects phosphopeptide, recognizes auto-phosphorylated protein, and recognizes endogenous protein from pervanadate-treated YT cells. ( a ) A mouse monoclonal pY841 JAK3 antibody was tested against increasing concentrations (1 ng, 10 ng, 100 ng and 1000 ng) of non-phospho-Y841 peptide (top) vs. phospho-Y841 peptide (bottom). A representative blot from n = 2 independent experiments is shown. ( b ) In vitro kinase assay −/+ 100 µM of ATP (lane a,b) with immunoprecipitated JAK3 from transfected Hek293 cells. Western blot with anti-pY841 JAK3 antibody (top panel), total anti-pY antibody (middle panel) and reblot for JAK3 (bottom panel). ( c ) IL-2 stimulated YT cells without (lane a,b) or with (lane c,d) pervanadate (PV), and then immunoprecipitated for JAK3. Western blot with anti-pY841 JAK3, total anti-pY and reblotted for JAK3. ( d ) IL-2 stimulation time course of 0–30 min (lanes a–h) in YT cells, immunoprecipitated for JAK3 and probed for anti-pY841 JAK3; total pY and JAK3 reblot used to confirm equal protein loading. All Western blot data shown above (b–d) are representative blots from n = 3 independent experiments.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Immunoprecipitation, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: JAK3 pY841 is upregulated in cells transformed by the JAK3 A573V variant and various T-cell lines. ( a ) HEK293 cells transfected with WT (lane a), K855A (lane b), M511I (lane c) or A573V (lane d) JAK3 were immunoprecipitated for JAK3, separated by SDS-PAGE and transferred to the PVDF. Membranes were Western blotted using anti-pY841 JAK3, anti-pY and reblotted for total JAK3 protein to confirm equivalent loading. Representative data from n = 3 replicates shown. The individual data points for replicate experiments are available in . ( b ) Densitometry analysis was performed using LI-COR Biosciences software. Anti-pY841 values were normalized against the total JAK3 protein and plotted as fold induction to WT JAK3. Error bars represent the mean +/− standard deviation from three independent experiments (n = 3). ( c ) The actively growing cell lines Hut78 (lane a), MT2 (lane b), Hut102 (lane c), Molt3 (lane d), HH (lane e), and SupT1 (lane f) were lysed and immunoprecipitated for endogenous JAK3. PVDF membranes were Western blotted with anti-pY841, anti-pY, or reblotted for total JAK3 to confirm the presence of protein. Representative data from n = 2 are shown.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Transformation Assay, Variant Assay, Transfection, Immunoprecipitation, SDS Page, Western Blot, Software, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: pH dependence of the folding energy for phosphorylated and non-phosphorylated Y841-JAK3. The folding energy values for non-phosphorylated Y841 (Y841-JAK3) and phosphorylated Y841 JAK3 (pY841-JAK3) were determined using PROPKA3, as shown by Equation (1) in Methods. Data points represent the difference between the kcal/mol values at the designated pH and the determined binding energy at pH = 0. A positive folding energy is indicative of a higher folding energy requirement compared to pH = 0, and negative values are indicative of a lower energy requirement compared to pH = 0.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: Structure of the JH1 kinase domain of JAK3 and surface electrostatic potential surrounding Y841. ( a ) View of the JAK3 JH1 kinase domain ribbon structure (amino acids 815-1124) is shown depicting Y841 (shown in red) located on the N-lobe. Positively and negatively charged surface regions are colored in blue (1 kT/e) and red (−1 kT/e), respectively. The Y841 surface region is circled in green for ( b ) non-phosphorylated Y841 kinase and ( c ) phosphorylated Y841 kinase.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: Dimerized structure of pY841 JAK3 determined using ZDOCK Server. ( a ) Structure of the pY841 kinase domain (amino acids 815-1124) dimer with Y841 depicted (red). ( b ) The electrostatic surface potential of the JH1 dimer depicted in ( a ). ( c ) The electrostatic surface potential for the binding interface of ( b ) opened by 90°. Green arrows depict binding pairs with the positive surface pointing to the corresponding negative surface of the dimer interface. Green circle encompasses the area containing the phosphorylated Y841 residue. ( d ) Depicted dimer in ( b ) was separated by 15 Å to show the electrostatic field lines between the interface. ( e ) Magnified image of kinase domain interface and electrostatic field lines in the cutout region.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Binding Assay, Residue

Journal: International Journal of Molecular Sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation

doi: 10.3390/ijms241511928

Figure Lengend Snippet: Partial sequence alignments for tyrosine and serine/threonine kinases found to possess a homologous Y residue at the positionally conserved JAK3 Y841 site. The partial JAK3 sequence (qlgkgnfgsvelcrYdplgdntgalvavk) containing Y841 (centered) was aligned with the full-length sequence for 95 kinases from the human kinome using the ClustalW2 Pairwise Sequence Alignment Tool (EMBL-EBI). Shown are the partial alignments for the JAK3 peptide containing Y841 aligned with homologous regions of human Lck (Y264), Lyn (Y267), Hck (Y282), Blk (Y260), Zap70 (Y231), PDGFR-B (Y686), VEGFR1 (Y876), VEGFR3 (Y1009), Flt3 (Y498), and ERK2 (Y43). Homologous Y residues are highlighted within individual kinase alignments. (*) indicates fully conserved residues, (:) indicates conservation between groups of strongly similar properties, and (.) indicates conservation between groups of weakly similar properties.

Article Snippet: The membranes were allowed to dry, re-activated using methanol and then incubated with 1% BSA blocking buffer (20 mM Tris, 137 mM NaCl, 0.25% Tween-20, 1% Bovine Serum Albumin (BSA)) for 1 h at room temperature, followed by incubation with the newly generated phosphospecific monoclonal antibody anti-pY841 JAK3 (GenScript, 1:5000), as described previously [ ].

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Growth-regulated Hsp70 phosphorylation regulates stress responses and prion maintenance

doi: 10.1101/759241

Figure Lengend Snippet: (A) Alignment of Hsp70 protein sequences in the region surrounding S151 in the NBD in prokaryotic and eukaryotic cells as indicated. (B) The S151 residue of Ssa1 in the NBD is located close to the interaction site with the SBD in the ATP-bound state. Left panel: ATP hydrolysis and nucleotide exchange is postulated to regulate structural conformation changes in Hsp70 proteins. In the ADP-bound state, the NBD (green) is in an open configuration, connected to the SBD (red: alpha-helical lid; blue: beta-sheet pocket) via a flexible linker. In the ATP-bound state, NBD and SBD undergo a conformational change to interact in a closed configuration. Right panel: In DnaK, A149 and D148 of the NBD are in close contact with K452 and E442 of the SBD (PDB: 4B9Q) ( Kityk et al , 2012 ). (C) GFP-Flag-Ssa1 (WT) or GFP-Flag-Ssa1 S151A (S151A) were expressed in a ssa1 ssa2 ssa3 ssa4 deletion ( Δssa1-4 ) strain. GFP-Flag-Ssa1 was isolated by immunoprecipitation and analyzed by quantitative Western blotting with anti-phospho-Ssa1 S151 and anti-Flag antibodies. (D) Total lysates from Δssa1-4 yeast cells expressing Flag-Ssa1 (WT), Flag-Ssa1 S151A (S151A), or Flag-Ssa1 S151D (S151D) were analyzed by wester blotting for Flag or ADH1 as a loading control. (E) Δssa1-4 yeast cells expressing Flag-Ssa1 (WT), Flag-Ssa1 S151A (S151A), or Flag-Ssa1 S151D (S151D) were spotted in fivefold serial dilutions and exposed to 30 or 39°C for 48 or 72 hr. (F and G) Growth of Δ ssa1-4 yeast cells expressing Flag-Ssa1 (WT), Flag-Ssa1 S151A (S151A), or Flag-Ssa1 S151D (S151D) was monitored at 30°C or 39°C as indicated. Growth curve is measured in log phase (F) and stationary (G) phase by OD600. 3 biological replicates were performed and error bars represent standard deviation. (H) Δ ssa1-4 yeast cells with an integrated HSE-YFP reporter and expressing Flag-Ssa1 (WT), Flag-Ssa1 S151A (S151A), or Flag-Ssa1 S151D (S151D) were exposed to varying temperatures as indicated for 30 min. YFP signal was measured by flow cytometer. 3 biological replicates were performed with at least 10,000 cells per measurement; error bars represent standard deviation.

Article Snippet: The S151 phospho-specific antibody was produced by PhosphoSolutions using a peptide containing the phosphorylated site.

Techniques: Residue, Isolation, Immunoprecipitation, Western Blot, Expressing, Control, Standard Deviation, Flow Cytometry

Journal: bioRxiv

Article Title: Growth-regulated Hsp70 phosphorylation regulates stress responses and prion maintenance

doi: 10.1101/759241

Figure Lengend Snippet: (A) Δ ssa1-4 yeast cells expressing GFP-Flag-Ssa1 (WT), or GFP-Flag-Ssa1 S151A (S151A) as well as galactose-inducible HA-His-tagged Cdk1 (Gal-yCDC28-HA-His) were grown in glucose (Glu) or galactose (Gal). Ssa1 was isolated by immunoprecipitation and analyzed by Western blotting with anti-phospho-Ssa1 S151, anti-His and anti-Flag antibodies. (B) Cdk1-as1 cells expressing GFP-Flag-Ssa1 (WT), or GFP-Flag-Ssa1 S151A (S151A) were treated with NM-PP1 (10 uM) for 3 hr. Ssa1 was isolated by immunoprecipitation and analyzed by Western blotting with anti-phospho-Ssa1 S151 and anti-Flag antibodies. (C) Recombinant Flag-tagged Ssa1 (WT) or Ssa1 S151A (S151A) was incubated with HA-His-tagged Cdk1 isolated from ssa1-4 Δ yeast cells expressing galactose inducible HA-His-tagged Cdk1 (Gal-yCDC28-HA-His) treated with raffinose (Raf) or galactose (Gal). Phosphorylation was analyzed by Western blotting with anti-phospho-Ssa1 S151 and Flag antibodies. Arrow indicates phosphorylated species. (D) Δ ssa1-4 yeast cells expressing GFP-Flag-Ssa1 (WT), or GFP-Flag-Ssa1 S151A (S151A) were treated with TORin (2 μM or 0.5 μM) for 30 min. Ssa1 was isolated by immunoprecipitation and analyzed by Western blotting with anti-phospho-Ssa1 S151 and anti-Flag antibodies. (E) Δ ssa1-4 yeast cells expressing GFP-Flag-Ssa1 (WT), or GFP-Flag-Ssa1 S151A (S151A) were treated with rapamycin (200 ng/mL) for 30 min. Ssa1 was isolated by immunoprecipitation and analyzed by quantitative Western blotting with anti-phospho-Ssa1 S151 and anti-Flag antibodies. (F) Δ ssa1-4 yeast cells expressing Flag-Ssa1 (WT), Flag-Ssa1 S151A (S151A), or Flag-Ssa1 S151D (S151D) were monitored for growth over time in the absence or presence of rapamycin (100 ng/mL). (G) Δ ssa1-4 yeast cells expressing Flag-Ssa1 (WT), Flag-Ssa1 S151A (S151A), or Flag-Ssa1 S151D (S151D) were spotted in fivefold serial dilution on control or rapamycin plates (100 ng/mL) as indicated.

Article Snippet: The S151 phospho-specific antibody was produced by PhosphoSolutions using a peptide containing the phosphorylated site.

Techniques: Expressing, Isolation, Immunoprecipitation, Western Blot, Recombinant, Incubation, Phospho-proteomics, Serial Dilution, Control

Journal: bioRxiv

Article Title: Growth-regulated Hsp70 phosphorylation regulates stress responses and prion maintenance

doi: 10.1101/759241

Figure Lengend Snippet: (A) V5-Hsc70 (WT) or V5-Hsc70 S153A (SA) were expressed in U2OS cells with concurrent Hsc70/Hsp70 depletion. Hsc70 was isolated by immunoprecipitation and analyzed by Western blotting with anti-phospho-Ssa1 S151 and anti-V5 antibodies. (B) U2OS cells expressing V5-Hsc70 (WT), V5-Hsc70 S153A (S153A), or V5-Hsc70 S153D (S153D) were transfected with control siRNA (siCON) or siRNAs directed against HSPA8(HSC70) and HSPA1A(HSP70)(siBoth) and seeded in 96-wells plates. Cells were treated with doxycylin to induce recombinant Hsc70 expression and incubated at 39°C for 24 hr. Cell viability was measured by MTT assay (Thermo Fisher Scientific). 3 biological replicates were performed and error bars represent standard deviation. * indicates p < 0.05 by student’s 2-tailed T test. (C) mCherry-tagged WT, S153A, or S153D Hsc70 was expressed in U2OS cells expressing Halo-Fibrillarin and also treated with the JF502 halotag ligand. Cells were exposed to heat shock (43°C) for 60 min and analyzed by fluorescence microscopy. White arrows indicate nucleolar Hsc70. (D) Quantification of results from (C) showing percentage of cells with overlap between mCherry-Hsc70 and Halo-Fibrillarin. *** indicates t test p value < 0.0001 comparing S153D to either wild-type or S153A.

Article Snippet: The S151 phospho-specific antibody was produced by PhosphoSolutions using a peptide containing the phosphorylated site.

Techniques: Isolation, Immunoprecipitation, Western Blot, Expressing, Transfection, Control, Recombinant, Incubation, MTT Assay, Standard Deviation, Fluorescence, Microscopy