Phospho protease inhibitor cocktail

NIH-3T3-TrkB-expressing cells were plated in 12-well plates at 100,000 cells/well and the following day were serum deprived for 6 h. Compound treatments to assess TrkB activation were performed at 1 μM for 20 min, while BDNF was used as a positive control at 500 ng/mL. Lysis was performed using a lysis buffer on ice (Thermo Scientific Pierce IP Lysis Buffer) with a Millipore phospho-protease inhibitor cocktail by Millipore for 10 min. After adding the loading buffer (5× Laemni) to 25 μg of the protein sample, samples were incubated at 95 °C for 5 min and subjected to SDS-PAGE. Nitrocellulose membrane transfer was performed at 350 mA for 2 h, followed by blocking at room temperature with 5% bovine serum albumin (BSA) for 1 h and primary antibody incubation in blocking solutions at 4 °C overnight and detection with HRP-conjugated secondary antibodies. ECL solution was used to detect chemiluminescence. Primary antibodies used are Phospho-TrkB (Tyr816) Millipore # ABN1381 and Anti-TrkB Abcam #ab33655, β-Actin.

Cells were plated in 12 well plates, at a density of 100,000 cells per well, with 6 h of serum deprivation carried out on the following day. To assess the effect of compounds on TrkB phosphorylation, 20-min treatments were applied, using either BDNF at 500 ng/mL (Peprotech 450-02) (positive control) or compounds at 1 μM.
Cell lysis for 10 min was carried out on ice using the Pierce IP Lysis Buffer by Thermo Scientific and a phospho-protease inhibitor cocktail by Millipore. After adding loading buffer (5 × Laemni), 25 μg from each protein sample were incubated for 5 min at 95°C and subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane at 350 mA for 2 h. Membrane blocking was performed using 5% bovine serum albumin (BSA) at room temperature for 1 h, before adding the primary antibodies to the blocking solution at 4°C overnight. HRP-conjugated secondary antibodies were used for the detection of chemiluminescence with ECL solution. Primary antibodies: Phospho-TrkB (Tyr816) Millipore # ABN1381, and Anti-TrkB Abcam #ab33655.

The cells were treated with BDNF at 500 ng/mL (positive control) or compounds at 1 μM for 20 min. After the compound treatment, the cells were plated in 12-well plates at 100,000 cells/well and were serum deprived for 6 h on the following day.
The cells were lysed on ice for 10 min in Pierce IP lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA), using a phospho-protease inhibitor cocktail by Millipore. A loading buffer (5× Laemni) was added to 25 μg from each protein sample, followed by incubation at 95 °C for 5 min and SDS-PAGE electrophoresis. The proteins were transferred to a nitrocellulose membrane for 2 h at 350 mA. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated with the primary antibodies in blocking solutions at 4 °C overnight before detection with HRP-conjugated secondary antibodies. Chemiluminescence was detected with ECL solution.
Primary antibodies used: Phospho-TrkB (Tyr816) Millipore # ABN1381 (Merck Millipore, Burlington, MA, USA), Anti-TrkB Abcam #ab33655 (Abcam plc., Cambridge, UK), Phospho-Akt (Ser473) CST #9271, Akt CST #9272 (Cell Signaling Technology, Danvers, MA, USA), GADPH Sigma # G8795 (Sigma-Aldrich, St. Louis, MO, USA) [18 (link)].