Alexa fluor 680

The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.: 1:1000; endocytosis assay: 1:25), antibodies against GluN2A (alomone labs; 1:500) and GluN1 (Synaptic Systems; 1:200), anti pGluN2BTyr1472 antibody (AAT Bioquest; WB 1:500); mouse (ms) antibodies against GluN2B (NeuroMab; WB 1:500), PSD-95 (NeuroMab; ICC 1:1000), Map2 (Sigma-Aldrich; ICC 1:2000), β3-tubulin (Synaptic System; WB 1:1000); rat antibody against β1-integrin CD29 (BD Pharmingen; 1:25).
Fluorescently labelled secondary antibodies that were used for ICC against rabbit, mouse, guinea pig were purchased from Invitrogen conjugated with either Alexa 488, 568, 647 (1:1000) or from Dianova conjugated with Cy3, Cy5 (1:1000). Fluorescently labelled secondary antibodies against ms, rb and guinea pig for quantitative immunoblotting were purchased from Invitrogen (ms Alexa Fluor 680, 1:20,000) and from Rockland (rb IRDye 800 W, 1:20,000).
Hyaluronidase (Hya, Sigma-Aldrich) was used at 100 units/ml, and TTX (0, 5 µM), CNQX (5 µM), Biccuculine (BCC) (10 µM), AP5 (10 µM), Ifenprodil (3 µM) was purchased from Tocris. All other chemicals and drugs were purchased from Sigma-Aldrich (USA).

Cancer cells treated with NPs encapsulating ELTN, FDTN, or a combination therefore were lysed with the lysis buffer [50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% NP40, and 5 mM EGTA containing 50 mM sodium fluoride, 60 mM β-glycerol-phosphate, 0.5 mM sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, and 10 μg/mL leupeptin]. Protein concentration was measured using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, United States). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins. The SDS-polyacrylamide gels were transferred onto PVDF membranes (Millipore, Bedford, MA, United States). The membranes were incubated with specific primary antibodies against JAK2, phospho-JAK2 (p-JAK2), STAT3, p-STAT3, Survivin (Cell Signaling Technology, San Diego, CA, United States), and subsequently with HRP-conjugated secondary antibodies conjugated to Alexa Fluor 680, or IRdye 800 (Rockland Immunochemicals, Inc. Gilbertsville, PA, United States). The intensities of the protein bands were scanned using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, United States).

Protein extracts were diluted with loading buffer and separated by electrophoresis on 8%-10% SDS-polyacrylamide gels before transfer to nitrocellulose membranes (Bio-Rad). The membranes were blocked in Odyssey blocking buffer (LI-COR Bioscience, Lincoln, NE) at room temperature for 1 hr, then incubated at 4°C overnight with primary antibodies: Cat S (1:1000), LC-3 (1:1000), GAPDH (1:3000). The membranes were washed 3 times in TBST and incubated with fluorescent secondary antibodies (Alexa Fluor 680 or IRDye 800, Rockland Immunochemicals, Gilbertsville, PA, US) for 1 hr at room temperature at 1:5000, blots were analyzed with the Odyssey infrared imaging system and Odyssey software.

Total cellular protein and nucleic protein were prepared as previously described [17] (link). Protein concentration was determined using the BCA Protein Assay kit (Pierce). Proteins (30 µg) were subjected to 10% SDS polyacrylamide gel electrophoresis and subsequently transferred onto nitrocellulose membranes (Pall). After washing with 0.1% TBS-T, membranes were incubated for 1 h at room temperature in blocking buffer (5% nonfat milk in TBS-T) and then incubated with appropriate antibodies (1∶500 dilution, NF-κB from Cell Signaling, others are from Santa Cruz) overnight at 4°C. After washing with TBS-T, they were then incubated with the fluorescently-labeled secondary antibodies conjugated with Alexa Fluor 680 or IRdye 800 (1∶10000, Rockland Immunochemicals, Gilbertville, PA, USA) for 1 h at room temperature. The membranes were then washed with PBS containing 0.1% Tween-20 and scanned with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) by setting the detection channels at 700 nm (for Alexa Fluor 680) and 800 nm (for IRdye 800CW). Scanned bands were quantified using the Odyssey software.