s151 phospho-specific antibody Search Results


s151 phospho-specific antibody  (PhosphoSolutions)
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PhosphoSolutions s151 phospho-specific antibody
(A) Alignment of Hsp70 protein sequences in the region surrounding <t>S151</t> 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.
S151 Phospho Specific Antibody, supplied by PhosphoSolutions, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(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.

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

(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.

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

(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.

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