EUROSCARF collection of haploid non-essential kinase deletes in BY4741 (MAT a; Δhis3-Δ1; leu2-Δ0; met15-Δ0; ura3-Δ0) background (www.uni-frankfurt.de/fb15/mikro/euroscarf/ ) was used to screen for S25 phosphorylation (Table S1 ). These stains were transformed with yHsp90His6-Ycplac111. The wild type BY4741 was also transformed with the same construct as well as the S25A mutant. These mutants were grown in liquid DO media with appropriate amino acids in 96-well plate. Cells were centrifuged at 4000 RPM for 5min and then washed with ice-cold TBS. Pellet were then resuspended in 50μL of protein loading buffer (10% SDS, 500Mm DTT, 50% Glycerol, 500mM Tris-HCL and 0.05% bromophenol blue dye) and then boiled for 5min followed by centrifugation at 4000 RPM for 5min. The lysate was spotted on nitrocellulose membrane 10 times and then probed with either anti-6x-His (ThermoFisher Scientific) or anti-phospho-(Ser/Thr) Phe (Cell Signaling) antibodies.
Anti phospho ser thr phe
Manufactured by Cell Signaling Technology
Sourced in China
Anti-phospho-(Ser/Thr) Phe is a laboratory product that detects phosphorylation of serine or threonine residues followed by a phenylalanine. It is used as a research tool in cell signaling studies.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using anti phospho ser thr phe
1
Screening Kinase Deletion Mutants for S25 Phosphorylation
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted using a protein extraction kit (#P0013C, RIPA Lysis Buffer, Beyotime). The protein concentration was measured using the BCA method (Biosharp BL521A). Equal amounts of proteins (30 μg) were separated and transferred to PVDF membranes. Following encapsulation in 5 % skim milk, the PVDF membranes were incubated with the following primary antibodies: anti-IRF1 (1:1000, #a7692, ABclonal, Wuhan, China), anti-phospho-(Ser/Thr) Phe (1:1000, Cell Signaling Technology, #9631), anti-STAT3 (1:1000, #10253-2-ap, Proteintech, Wuhan, China), anti-phospho-STAT3-Y705 (1:200, #AP0070, ABclonal), anti-PD-L1 (1:1000, #A1645, ABclonal), and GAPDH (1:50000, #60004-1-Ig, Proteintech). Following incubation with secondary antibodies (#BL003A/#BL001A, Biosharp, Beijing, China), an enhanced chemiluminescence (ECL) system was used to visualize the protein bands, followed by quantification with ImageJ software.
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