Halttm phosphatase inhibitor cocktail

pVEGFR2 activation was assessed by Western blot analysis, a valuable method for evaluating endothelial cell activation. HuVECs and LECs were starved for 2 h and then treated for 20 min with VEGFC (100 ng/mL) in the presence or absence of 1E9 antibodies (10 µg/mL). Cells were lysed in Laemmli buffer containing a 2% SDS, 10% Glycerol, 60 mM Tris-HCl, and 1× Halt TM phosphatase inhibitor cocktail (Thermo Fischer, Illkirch, France). DNA was fragmented by sonication. Lysates supplemented with 0.002% bromophenol blue and 100 mM DTT were heated at 96 °C, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, Burlington, Massachusetts, United States). Membranes were probed with the following antibodies: pVEGFR2 (Tyr1175), CST, 2478S; VEGFR2, CST, 2479S; and β-actin (D6A8) CST, 8457.

ApoE protein expression was detected in neuronal lysate and astrocyte and neuron conditioned media by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot. Medium was collected and centrifuged at 10,000 × g for 10 min at 4°C before use. Neuronal lysate was washed in PBS, homogenized/lysed in a buffer containing 20 mM Tris, 150 mM KCl, 5 mM MgCl₂, and 1% NP40 with Halt^TM Protease inhibitor cocktail (Thermo Fisher Scientific, 78430) and Halt^TM Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78428), and centrifuged at 20,000 × g for 20 min at 4°C. The supernatant, which contained the intracellular proteins, was further used for Western blot. The media and lysate samples were mixed with Novex NuPage LDS sample buffer (Invitrogen, NP0007) and NuPage sample reducing agent (Invitrogen, NP0004), heated for 10 min at 70°C and loaded on a NuPAGE^TM 4–12% Bis-Tris protein gel (Invitrogen, NP0321). Secreted proteins in medium and intracellular proteins in lysate were separated by SDS PAGE. SeeBlue^® Plus2 pre-stained protein standard was used as a molecular weight marker.

Proteins were extracted from the collected cells as previously described (Menon and Beningo, 2011 (link)). Briefly, triple-detergent lysis buffer (TDLB; 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) was mixed with Protease Inhibitor Cocktail (Sigma) and Halt^TM Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The cell pellet was incubated with the lysis buffer solution for 20 min under ice-cold conditions. The solution was then centrifuged at 4°C for 10 min to isolate protein from cell debris. Protein concentration was determined by the DC protein assay (Bio-Rad).

HeLa cells were cultured in DMEM Glutamax I (GIBCO) (a Ca²⁺-containing medium) supplemented with 10% fetal bovine serum (GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO) at 37 °C in a 5% CO₂ humidified atmosphere. For transfection, cells were plated onto sterile 10 mm coverslips coated with 20 μg/ml poly-L lysine (Sigma) or 6-well tissue culture plates and transiently transfected with Lipofectamine^®2000 (Invitrogen), as per the manufacturer’s protocol. Cell viability was reduced in cultures expressing either the DI and DII pores (data not shown).
To prepare lysates for Western blotting, cells were harvested by scraping, and then lysed in Ripa buffer (150 mM NaCl, 50 mM Tris, 0.5% (w/v) sodium deoxycholic acid, 0.1% (v/v) sodium dodecyl sulphate, 1% (v/v) Triton X-100, pH7.4), supplemented with EDTA-free protease inhibitors (Roche) and Halt^TM phosphatase inhibitor cocktail (Thermo Scientific) for 30 min on ice. Samples were centrifuged at 15,000 g at 4 °C for 15 min and the resulting supernatants stored at −20 °C. Protein concentrations were calculated using a bicinchoninic acid assay, calibrated to bovine serum albumin protein standards.

DMEM/F12 (F12), Trypsin-EDTA, Igepal CA-930, Akt-IV, and SB216763 were purchased from Sigma–Aldrich, Inc. (St. Louis, MO, USA). Fetal calf serum (FCS) was acquired from Equitech-Bio, Inc. (Kerrville, TX, USA). A cocktail of sodium penicillin G, streptomycin sulfate, and amphotericin B was purchased from Gibco-BRL (Gaithersburg, MD, USA). Halt-TM Phosphatase inhibitor cocktail was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Protease inhibitor cocktail was acquired from GE Healthcare Biosciences (Little Chalfont, UK). Trizol reagent and EXPRESS One-Step SYBR GreenER Universal Kit were purchased from Invitrogen (Carlsbad, CA, USA). Bovine IL-10 and IL-8 TSZ ELISA kit were purchased from Biotang (Waltham, MA, USA). Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969). The generation of pCF empty vector was done by the enzyme digestion of pCF CREB M1 with SacI restriction enzyme, and then the purification and ligation of the 5-kb fragment were generated.

Cells and isolated EVs were lysed in RIPA buffer (100mM Tris-HCl pH8, 300 mM NaCl, 2% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with Complete Ultra Mini EDTA-free protease inhibitor mixture (1 tablet/10 ml, Roche Applied Science) and HaltTM phosphatase inhibitor cocktail (Thermo Fisher Scientific). 30 μg protein extracts were resolved on a SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with antibodies recognizing CD63 (sc-5275, Santa Cruz Biotechnology; 0.4 μg/ml) or actin (clone C4, MAB1501, Millipore; 1:500). The blots were developed using IRDye800CW-conjugated goat anti-mouse secondary antibody (926–32210, LI-COR; 1:7000) and the Odyssey IR Imaging system.