Protease phosphatase inhibitor

Left brain hemispheres from mice in the ^99mTc-albumin experiments were immediately flash frozen in liquid nitrogen and stored at −80 °C for ten ^99mTc-albumin half-lives (~60 h). Hippocampus and striatum were dissected and sonicated in radioimmunoprecipitation assay (RIPA) buffer with protease/phosphatase inhibitors (Thermo; Rockford, IL 1:100). Samples were then centrifuged for 15 min at 12000 × g at 4 °C and supernatant collected and standardized with a bicinchoninic acid (BCA) assay. Brain extracts of equivalent total protein content were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20 µg total protein/lane) under reducing conditions, transferred to nitrocellulose, and probed with rabbit polyclonal claudin-5 (Invitrogen, Eugene, OR; 1:1000) and rabbit monoclonal β-actin (Cell signaling, Danvers, MA; 1:10000) as loading controls. Blots were then probed with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch; 1:5000) and visualized by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). Optical densities of the immunoreactive protein bands were quantified using Image-Quant with background subtraction and normalization to loading control.

We conducted the experiment in accordance with the procedure outlined by Bassani-Sternberg et al. [8 (link)]. Briefly, we lysed a single pellet of 3E7 SK-OV-3 cells with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease/Phosphatase inhibitors (Thermo), 1 mM PMSF, and 1% octyl-β-D glucopyranoside (Sigma) in PBS at 4 °C for one hour. The lysate was cleared for one hour at 20,000 x g prior to immunoaffinity purification of HLA class I molecules with the cross-linked W6/32 antibody. We then washed beads with 10 x bead volume of 150 mM NaCl, 20 mM Tris.HCl (buffer A), 10 volumes of 400 mM NaCl, 20 mM Tris.HCl, 10 volumes of buffer A again, and lastly with seven volumes of 20 mM Tris.HCl, pH 8.0. Next, we eluted HLA class I molecules by the addition of 500 μl of 0.1 N acetic acid at room temperature in two steps following a five-minute incubation each time.

For Western blots, cells were grown to 70% confluence in 6-well plates. Cells were then treated with a vehicle (0.1% DMSO) or TKA001 for 2.5 h. Cells were then placed on ice, washed with cold phosphate-buffered saline (PBS), then harvested in 60 µL of cold RIPA buffer containing protease/phosphatase inhibitors (Thermo Fisher Scientific). Cells were incubated on ice for 20 min, then centrifuged at 20,000× g for 10 min, and supernatants were collected. Supernatants were standardized to a concentration of 2 mg/mL and combined with 6× loading buffer containing SDS, glycerol, beta-mercaptoethanol, and bromophenol blue. Samples were boiled at 100 °C for 10 min, and 15 µL was loaded on a Tris/glycine SDS-polyacrylamide gel. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and immunoblotted with indicated antibodies. The primary antibodies used were pS6 (S240/244): CST 5364, p-AKT (S473): CST 4060, ATK: CST 4691, and Vinculin: CST 13901. The secondary antibody was HRP-linked anti-rabbit IgG (CST 7074). Immunoblots were imaged using an enhanced chemiluminescent detection kit (ECL, Bio-Rad, Hercules, CA, USA) and visualized on a LiCor Odyssey Fx Imaging System (LiCor, Lincoln, NE, USA).

Cells were harvested by scraping, washed in DPBS lacking calcium and magnesium (Gibco, 14190-144), and lysed in RIPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1% Tween-20) containing protease/phosphatase inhibitors (Thermo Scientific). Total protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to PVDF membrane, and blocked in 5% milk solution. The following antibodies were used: goat anti-human/mouse anti-Bcl-2 (AF810, R&D Systems, Minneapolis, MN, USA) and mouse anti-human anti-puromycin (MABE343, Millipore Sigma, St. Louis, MO, USA).