Dylight fluors conjugated

Cells were washed with ice-cold PBS and lysed with RIPA buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholatemonohydrate, 0.1% SDS, supplemented with protease (Roche) and phosphatase inhibitors cocktails (Sigma Aldrich)]. Cells were incubated on ice for 30 min. Mice tissue samples were lysed in RIPA buffer, centrifuged at 16,100 g for 10 min and the protein concentration of supernatants was determined using a DC Protein Assay (Bio-Rad). Tissue and cell lysates were then mixed with 2x Laemmli buffer with b-mercaptoethanol, boiled at 100 C for 5 min and resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk or 3% BSA in PBS, incubated with primary antibodies overnight, followed by HRP-conjugated (GE Healthcare) or DyLight Fluors-conjugated (Invitrogen) secondary antibodies. Immunoreactive bands were visualized with an ECL detection kit (Pierce, Thermo Scientific) in Bio-Rad ChemiDoc Imager or with direct infrared fluorescence detection using LICOR-Odyssey apparatus. Densitometric analysis on the immunoblots was performed using ImageJ program or IMAGE STUDIO Lite software. Western blots presented in each figure are representative of at least three biological replicates.

Cells were washed with ice-cold PBS and lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholatemonohydrate, 0.1% SDS, supplemented with protease (Roche) and phosphatase inhibitors cocktails (Sigma Aldrich)). Cells were incubated on ice for 30 min. Indicated cell lysates were pre-treated with Lambda phosphatase (100 Units/125 mg lysates) in 30 C for 1 h. Tissue and cell lysates were then mixed with 2x Laemmli buffer with b-mercaptoethanol, boiled at 100 C for 5 min and resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk or 5% BSA in PBS, incubated with primary antibodies overnight, followed by HRP-conjugated (GE Healthcare) or DyLight Fluors-conjugated (Invitrogen) secondary antibodies. Immunoreactive bands were visualized with an ECL detection kit (Pierce, Thermo Scientific) in Bio-Rad ChemiDocÔ Imager or with direct infrared fluorescence detection using LICOR-Odyssey apparatus. Densitometric analysis on the immunoblots was performed using ImageJ program or IMAGE STUDIO Lite software.