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Western blot using IRS1 (phospho S307) polyclonal antibody (Cat # PAB10078) shows detection of a band at ~180 KDa believed to represent phosphorylated IRS1 (arrowhead).
Lane 1 shows staining of human 293 cell lysate.
Lane 2 shows staining of 293 cell lysate prepared from cells serum-starved for 18 h followed by treatment with 5 ug/mL of anisomysin for 30 min.
The pronounced staining of the band at 180 KDa is not seen when the antibody was pre-incubated with immunizing peptide prior to reaction (data not shown).
The identity of the intensely reactive bands at ~70 kD in both lane 1 and 2 is unknown, although these bands were also competedout by pre-incubation with the immunizing peptide.
Approximately 25 ug of each lysate was separated on a 4-20% Tris-Glycine gel by SDS-PAGE and transferred onto nitrocellulose.
After blocking with 5% goat serum, 0.5%BLOTTO in PBS, the membrane was probed with the primary antibody diluted to 1:250.
Reaction occurred overnight at 4°C followed by washes and reaction with a 1:10,000 dilution of IRDye™800 conjugated Gt-a-RabbitIgG [H&L] MX for 45 min at room temperature (800 nmchannel, green).
Molecular weight estimation was made by comparison to prestained MW markers in lane M (700 nm channel, red).
IRDye™800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR.
IRDye is a trademark of LI-COR, Inc.