Western blot analysis of extracts of various cell lines, using Phospho-MAP3K5-S83 antibody at 1:1000 dilution. K562 cell lysate were treated by CIP at 37℃ for 1 hour, treated by TNF-α for 30 minutes after serum-starvation overnight or treated by ATP at 30℃ for 1 hour.
Secondary antibody: HRP Goat Anti-Rabbit IgG at 1:10000 dilution.
Lysates/proteins: 25ug per lane.
Blocking buffer: 3% BSA.
Detection: ECL Basic Kit .
Exposure time: 60s.
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