Mouse anti rabbit igg hrp

Proteins were extracted from undifferentiated and RA-differentiated SH-SY5Y cells using RIPA Buffer and quantified using Bradford Assay (Bio-Rad, Hercules, CA, USA). After denaturation at 95 °C, 25 µg of protein for each sample was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Immobilon–P, Millipore, Burlington, MA, USA). Membranes were blocked with 5% skimmed milk in TBS for 1 h. The blots were incubated at 4 °C overnight with Anti-Tyrosine Hydroxylase (1:1000, Millipore, Temecula, CA, USA) and Anti-β-Actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). The blots were incubated for 1 h at room temperature with the following secondary antibodies: mouse anti-rabbit IgG-HRP (1:850, Santa Cruz Biotechnology, Dallas, TX, USA) and Chicken anti-Mouse IgG Secondary Antibody, HRP (1:1000, ThermoFisher, Rockgord, IL, USA). ChemiDocTM MP System (Bio-Rad) acquired bands after exposure to an enhanced chemiluminescence system (Luminata Western HRP Substrates, Millipore, Burlington, MA, USA).

Loading buffer was added to 10 µg of EV protein sample and boiled at 95 °C for 5 min, separated in a 12% SDS-polyacrylamide gel (SDS-PAGE) and electrophoretic transferred to nitrocellulose membranes. Membranes were blocked in 2.5% nonfat dry milk in 1x TBS-T (0.5% Tween-20), at room temperature and incubated with the following primary antibodies: rabbit anti-CD9 (1:2000) (sc-9148; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-CD10 (1:3000) (GTX111680; Genetex, Irvine, CA, USA), mouse anti-HSC70 (1:2000) (sc-7298; Santa Cruz Biotechnology, Inc.), mouse anti-TSG101 (1:1000) (sc-7964; Santa Cruz Biotechnology, Inc.). Membranes were then washed with 1x TBS-T (0.5% Tween-20). The secondary antibodies used were the mouse IgGκ BP conjugated to HRP (1:2000) (sc-516102; Santa Cruz Biotechnology, Inc.); and the mouse anti-rabbit IgG-HRP (1:2000) (sc-2357; Santa Cruz Biotechnology, Inc.). Secondary antibodies were incubated for 2 h, with agitation at room temperature. Following TBS-T washes, protein bands were detected using the chemiluminescence reagent WesternBright ECL HRP substrate (Advansta Inc., San Jose, CA, USA.) and images acquired with G:BOX Chemi XX9 gel imaging system (Syngene, Cambridge, UK).

Protein was isolated from endothelial cells with a RIPA Kit (Sigma Aldrich, R0278, Burlington, MA, United States) supplemented with protease and phosphatase inhibitors. Protein concentration was determined via BCA protein assay (Thermo Fisher Scientific, 23227, Oakwood, OH, United States) per manufacturer’s instructions. Protein lysates (40 μg/lane) were loaded for probing CXCR4 (1:500 dilution; Abcam, Ab181020, Waltham, MA, United States). Following primary antibody incubation (overnight at 4^°C), blots were incubated (1 h at room temperature) with a mouse anti-rabbit IgG-HRP (1:3,000 dilution; Santa Cruz, SC-2357, Dallas, TX, United States). Immunoreactive bands were detected using a western blot imaging system (Cytiva, Amersham ImageQuant 800, Marlborough, MA, United States). GAPDH was used as a loading control (1:400 dilution; Millipore Sigma, MAB374, Burlington, MA, United States).

The specific procedure in our published paper was followed.^{39 (link)} Brief description was as follows: cells at 5 × 10⁵ per well (six-well plate) were lysed as one amount of a sample. Equal amounts of protein extracts were separated on 10% SDS-polyacrylamide gel electrophoresis, and then transferred into a PVDF membrane (Millipore, Germany) at 200 mA for 2 h. PVDF membranes were blotted with 5% milk for 1 h and then the blots were probed overnight with primary antibodies (β-actin, 1:2 000, sc-47778; Cx43, 1:3 000, #11370; N-cadherin, 1:2000, #76057; Smad3, 1:3 000, #28379; Smad4, 1:5000, #40759; p-Smad3, 1:2 000, #52903; Abcam, Cambridge, UK), then added corresponding secondary antibodies (m-IgG_КBP-HRP, 1:4 000, sc-516102; mouse antirabbit IgG-HRP, 1:2 000, sc-2357, Santa Cruz) and incubated for 2 h. Signals from PVDF membranes were taken by a Western Blotting Luminol Reagent Kit (Santa Cruz).