Supplementary Figure 1.docx (114.05 kB)
Supplementary Figure 1: An assay of human tyrosine protein kinase ABL activities by using an Escherichia coli protein-expression system
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posted on 2021-04-06, 08:00 authored by E Kinoshita, Emiko Kinoshita-Kikuta, Momoka Yoshimoto, Marina Yano, Tohru KoikeDemonstration of the
presence of tyrosine-phosphorylated GST-Abltide in WT and H396 lysates by
western blotting with anti-phosphotyrosine antibody. E. coli BL21(DE3) was
co-transformed with pET21a(+)_ABL (WT, Y253F, E255K, T315I, M351T, or H396P)
and pCDF_GST-Abltide. The cells were suspended in a Tris-buffered saline (TBS,
pH 7.4) containing 1 mM dithiothreitol, and then they were sonicated. The
soluble fractions of lysate samples (200-µg protein per 100 µL) were separately
incubated with GST-Accept resins (each 100 µL) for 10 min at room temperature.
Each GST-Accept resin was washed with TBS containing 1 mM dithiothreitol and
was mixed with 3× sample-loading buffer for SDS-PAGE. All the samples were
analyzed by Phos-tag SDS-PAGE (10% w/w polyacrylamide and 15 µM Zn2+–Phos-tag)
followed by western blotting with anti-phosphotyrosine antibody PY20 (left-hand
panel). The same blot was re-probed with anti-GST antibody (right-hand panel).