Mouse anti gapdh

Thirty micrograms of whole cell lysates were reduced in a mixture of 1x LDS sample Buffer (TruPAGE, PCG3009) and 50 μM DTT at 80°C for 10 minutes. Reduced lysates were then ran on a 4–15% Criterion TGX Stain-Free Protein Gel (BioRad, 5678083) in 1x tris/glysine/SDS (BioRad, 1610732) at 150 v for 1 hour and transferred to a nitrocellulose membrane in 1x tris/glycine (BioRad, 1610734) containing 20% methanol for 1 hour. The membrane was then washed in 1x TBS-T and blocked for 1 hour in 5% BSA-TBS-T. Primary mouse anti-IκBα (Novus, NB100-56507), mouse anti-phospho-IκBα S32/36 (Cell Signaling Technology, 9246S), and mouse anti-GAPDH (Biolegend, 607902) was then diluted to 1 μg/mL in 5% BSA-TBS-T and incubated at 4°C overnight with agitation. The membrane was then washed 3x for 10 minutes per wash in 1x TBS-T and secondary goat anti-mouse HRP (Abcam, ab205719) diluted 1:1000 in 5% BSA-TBS-T was incubated at room temperature for 1 hour after which the membrane was washed 3x for 10 minutes per wash in 1x TBS-T and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34094) on a BioRad ChemiDoc MP Imaging System.