Odyssey infrared ir imaging system

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (pH 7.4) (50 mM Tris-HCl, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1% protease inhibitor mixture, 1 mM sodium orthovanadate, and 1 mM sodium fluoride). Proteins were separated on an SDS-PAGE gel and transferred onto nitrocellulose membranes, followed by blocking in 0.1% phosphate-buffered saline (PBS)–Tween (PBST) with 5% bovine serum albumin (BSA) and incubation with primary antibodies at 4°C overnight. Detection was performed with IRDye 800 CW-conjugated anti-rabbit IgG and IRDye 680 CW-conjugated anti-mouse IgG secondary antibodies (Li-Cor) or horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Immunoreactive bands were visualized using an Odyssey infrared (IR) imaging system (Li-Cor). The bands were quantified using Quantity One software (Bio-Rad).

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (pH 7.4) (50 mM Tris-HCl, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1% protease inhibitor mixture, 1 mM sodium orthovanadate, and 1 mM sodium fluoride). Proteins were separated on an SDS-PAGE gel and transferred onto nitrocellulose membranes, followed by blocking in 0.1% phosphate-buffered saline (PBS)–Tween (PBST) with 5% bovine serum albumin (BSA) and incubation with primary antibodies at 4°C overnight. Detection was performed with IRDye 800 CW-conjugated anti-rabbit IgG and IRDye 680 CW-conjugated anti-mouse IgG secondary antibodies (Li-Cor) or horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Immunoreactive bands were visualized using an Odyssey infrared (IR) imaging system (Li-Cor). The bands were quantified using Quantity One software (Bio-Rad).

Immunoblot analysis of MAP kinase activation was performed with a polyclonal antibody to a synthetic peptide derived from the C-terminus of p44/42 MAPK (Erk1/2) (#9102, CST), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb (#9106, CST), and IRdye-labelled secondary antibody and size-markers using the Odyssey Infrared (IR) imaging System (Li-Cor, Lincoln, NE, USA) according to the manufacturer’s protocol and as described by [68 (link),76 (link)].