Phosphostop phosphatase inhibitor

Cells were collected in 1X phosphate buffered saline (PBS), centrifuged and re-suspended in lysis buffer (1% Triton-X-100, 100 mM sodium chloride, 50 mM HEPES, 5% glycerol, protease inhibitor mixture and PhosphoSTOP phosphatase inhibitor (Roche Applied Science) as previously described [59 (link)]. Concentrations of whole cell lysate protein were measured by Bradford assay. Twenty micrograms of total protein samples were prepared in Laemmli buffer, boiled (100 °C; 5 min) and loaded into a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane, blocked with with 5% bovine serum albumin (BSA)/Tris buffered saline with Tween 20 (TBST) (1 h at RT) and incubated overnight with anti-c-Myc antibody (Cell Signalling Technology; 1:1000; 4 °C). Blots were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000, Cell Signalling Technology; 45 min; RT). Blots were stripped, blocked 5% BSA/TBST (1 h; RT) and re-probed with anti-GAPDH antibody (Santa Cruz Biotechnology; 1:3000; 40 min; RT) and anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000, Cell Signalling Technology; 45 min; RT). Protein was detected with enhanced chemiluminescent substrate.

Cultured primary neurons or HEK293T cells were washed with ice-cold PBS and lysed with RIPA buffer (10 mM Tris-HCl pH = 7.2, 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 5 mM EDTA, 0.25% Na-deoxycholate) supplemented with Complete protease inhibitor and PhosphoStop phosphatase inhibitor (Roche). For analysis of hippocampal tissue, the hippocampi from P10 mice were harvested and homogenized in RIPA buffer using a Dounce tissue homogenizer. Lysates were cleared by centrifugation and boiled in Laemmli sample buffer. Equal amount of total proteins were loaded. Samples were then analyzed by SDS-PAGE, transferred onto nitrocellulose membranes, probed with appropriate primary and HRP-labeled secondary antibodies and revealed by enhanced chemiluminescence.

Cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM Nonidet P-40, 1 mM sodium deoxycholate, 1 mM EDTA, and 1 mM sodium orthovanadate) containing complete protease inhibitor cocktail (Roche) and Phosphostop phosphatase inhibitor (Roche) at 4° C for 30 min as decribed.
Protein concentrations were determined by protein assay (Bio-Rad). Denatured protein samples were separated using 12% SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked with 5% dry milk in TBS/Tween and incubated with primary antibodies diluted in TTBS overnight at 4° C. Primary antibodies included YB-1 c-term (polyclonal rabbit; 1:1000; Mertens/Eurogentec); DbpA (polyclonal rabbit; 1:1000; Mertens/Eurogentec); Akt1 (monoclonal mouse; 1:1000; Cell Signalling #2967); pAkt(S473) (monoclonal rabbit; 1:1000; Cell Signalling #4058); pAkt(Thr308) (monoclonal rabbit; 1:1000; Cell Signalling #2965); HA-Tag (polyclonal rabbit, 1:1000; Santa Cruz #sc-805). Secondary HRP-conjugated goat anti-rabbit IgG (Biozol) or goat anti-mouse IgG (Biozol) antibodies were added for 60 minutes. Following additional washing steps SuperSignal chemiluminescence substrate (GE Healthcare; #RPN2232) was added and emitted light detected by Intas imaging system.

Whole-cell lysates were collected in lysis buffer supplemented with cOmplete ULTRA protease inhibitor (Roche) and Phospho-STOP phosphatase inhibitor (Roche). After protein quantities were normalized using BCA Protein Assay Kit (Thermo Scientific), samples were denatured with SDS loading dye at 95 °C for 5 min. When probing for NOXA, samples were resolved on a 15% polyacrylamide gel at 35 mA for 25 min. For all other proteins, samples were resolved on 4–15% or 10–20% TGX Criterion gradient gels (Bio-Rad) at 200 V for 35 min. Proteins were transferred to 0.2 µm nitrocellulose membrane at 100 V for 20 min (for NOXA) or 50 min (for all other proteins). The membrane was blocked with 5% milk in TBST for 45 min, washed with TBST, and incubated with primary antibodies at 4 °C for three nights (for NOXA) or overnight (for all other proteins). After washing with TBST, the membrane was incubated with secondary antibody at room temperature for one hour. Membranes were washed with TBST, and chemiluminescence reaction was performed using ECL Western Blotting Substrate (Pierce). Chemiluminescent films were exposed to the membrane in a darkroom and developed using Kodak X-OMAT 2000A.

Whole cell lysates were collected in CHAPS immunoprecipitation buffer supplemented with cOmplete ULTRA protease inhibitor (Roche) and Phospho-STOP phosphatase inhibitor (Roche). Protein concentration was measured using BCA Protein Assay Kit (Thermos). For each sample, 750 µg protein was aliquoted into two 1.5 mL Eppendorf tubes. Antibodies for the protein of interest and control IgG were incubated with the samples overnight at 4 °C on a rocker. Protein A or Protein G magnetic beads, depending on the species of the primary antibody, were added, and the samples were rocked for an additional 45 min at 4 °C on the rocker. The beads were precipitated three times using a magnetic stand apparatus and washed with fresh CHAPS buffer after each precipitation. After the third wash, SDS loading dye was added, and samples were boiled at 95 °C for 5 min. Samples were resolved by western blot.

Harvested cell pellets were lysed in 1xRIPA buffer supplemented with 1x EDTA-free cOmplete protease inhibitor (Roche) and 1x PhosphoSTOP phosphatase inhibitor (Roche) for 30 minutes on ice with two rounds of 15 second vortexing. Lysates were cleared at 21,000 ×g for 10mins at 4°C. Protein concentration was determined by BCA assay and BSA standard curve (Pierce), and samples were adjusted to 1μg/μL total protein with 1xRIPA and SDS-PAGE sample loading buffer was added (62.5 mM Tris-HCl (pH 6.8), 2.5% SDS, 0.1% bromophenol blue, 10% glycerol, 5% β-mercaptoethanol (v/v)). 10μg of total protein was loaded per lane onto a 4–20% Criterion Tris-HCl Protein gel (Bio-Rad) and separated by electrophoresis at 150 V for 1hr. Proteins were transferred and immobilized onto a nitrocellulose membrane (GE Healthcare) by electrophoresis for 1h at 100V in standard transfer buffer containing 20% methanol. Membranes were blocked for an hour at room temperature and then then probed overnight in a 1:1000 dilution of 1° antibody (unless otherwise indicated) at 4°C and in a 1:10,000 2° antibody at room temperature for 1hr at in the appropriate blocking buffer. Chemiluminescent and fluorescent signals were visualized with an Odyssey FC imager (LICOR).