Halt protease and phosphatase inhibitor cocktail 100x

Flash-frozen tissues were thawed and homogenized using standard methods. Briefly, tissue biopsies were cut into small pieces and placed in a snap cap tube with PBS, 10 μl/mL of Halt Protease and Phosphatase Inhibitor Cocktail 100X (Thermo Scientific, Waltham, MA), and 3.2 mm stainless-steel beads. Tubes were then homogenized with a TissueLyser II (Qiagen, Germantown, Maryland) run at 30 frequency for 2 minutes, three times. Samples were then centrifuged to remove any debris. The resulting supernatant was analyzed for protein concentration using the Micro BCA Protein Assay and read on the SpectraMax Plus 384 Microplate Reader (Molecular Devices). The samples were then frozen and stored at -80°C until assayed.

BDNF (PHC7074) and ToxBLAzer reagent (K1136, P36400) were obtained from Life Technologies. NGF-β (N1408), NT-3 (N1905), NT-4 (N1780), and Thapsigargin (T9033) were obtained from Sigma-Aldrich. Tool small molecules: LM22A-4, amitryptiline, 7,8-dihydroxyflavone and N-acetyl serotonin were supplied by CHDI and used after passing QC analysis; B_AG peptide was synthesized by Peptide Synthetics; tool antibody panel was sourced commercially (BD mAb #610102; Millipore pAb #07-225) or from Pfizer Inc (38B8 and 29D7 mAbs); a pan-Trk kinase inhibitor was synthesized at BioFocus [26] . All-trans retinoic acid (R2625) and BSA (A0281) were obtained from Sigma-Aldrich. PathHunter® detection kit (93-0001) was obtained from DiscoveRx. DMSO (D/4121/PB17), Triton-X100 (10102913) and Halt Protease and Phosphatase Inhibitor Cocktail (100X) (78444) were obtained from ThermoFisher Scientific. Recombinant pAKT1 (31145) for Meso Scale Discovery studies was obtained from Active Motif; whole cell lysate kit – Phospho (Ser473)/ Total AKT assay (K11100D-2) was obtained from Meso Scale Discovery.

HMC3 cells were seeded into a 6-well plate at 1 × 10⁶ cells/well in EMEM without FBS. After 1-hour incubation with designated treatments, the supernatant was removed, and cells were washed three times by DPBS. The cell lysate was obtained by lysing cells using NP-40 lysis buffer (1% NP40, 50 mM Tris-HCl, pH = 8, 150 mM NaCl) with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (ThermoFisher). After removing debris by centrifugation, the total protein amount normalized by Pierce BCA Protein Assay Kit (ThermoFisher). Protein samples were resolved by 10% SDS-polyacrylamide gels (Biorad) and later transferred onto Immun-Blot PVDF membranes (Biorad). Proteins were probed with specific primary antibodies and secondary antibodies diluted in 5% BSA TBST [21 (link), 24 (link), 29 (link)]. The primary antibodies used are: SYK (1:1000, Cell Signaling 13198S), Phospho-Syk (Tyr525/526) (1:1000, Thermo Fisher MA5-14918), β-Actin (1:1000, Cell Signaling 4970S), SHP1 (1:1000, Cell Signaling 3759S), Phospho-SHP-1 (Tyr564) (1:1000, Cell Signaling 8849S), and LILRB2 (1:1000, Thermo Fisher PA5-46983).
The immunoreactive bands were visualized with the West Pico PLUS Chemiluminescent Substrate (ThermoFisher). The immunoreactive bands were quantified using ImageJ. Three independent treatment replicates were conducted with the representative immunoblot shown.

Knock out cells were tested for the presence of CD9 and CD81 at the protein level using 15% SDS-PAGE and Western blotting. A549 cells were lysed with 100 µl lysis buffer (25 mM Tris–HCl pH 7.4 containing 150 mM NaCl, 1% sodium deoxycholate, 1% TritonX-100, 0.1% SDS and 1 mM EDTA and 5% glycerol) and 1 µl protease inhibitor (Halt protease and phosphatase inhibitor cocktail (100X) (Thermo Fisher, Waltham, MA, USA) at 4 °C for 30 min. The lysed cells were centrifuged at 15,000 × g at 4 °C for 15 min. The protein concentration of the supernatant was quantified using a BCA kit (Thermo Fisher, Waltham, MA, USA). Cell lysates were diluted with sample buffer and heated at 100 °C for 5 min. Thirty µg of protein was run on 15% SDS gels, transferred to a nitrocellulose membrane and probed with monoclonal anti-CD9 antibody (clone 602.29) or anti-CD81 antibody (clone 1D6, Bio-Rad, USA) at dilution of 1:1000. Rabbit anti-mouse IgG/HRP (Dako Cytomation, Denmark) at dilution of 1:2000 was used as secondary antibody. BM Chemiluminescence Blotting Substrate (Roche, Germany) was added on the membrane before exposure to an X-ray film.