Anti cxcr4

The following primary antibodies were employed: anti-VAMP1 (1:200, generated as described in [23 (link)]), anti-CXCR4 (Abcam, cat. Ab 124824, 1:400, Cambridge, UK), and anti-Neurofilament (Abcam, cat. Ab 4680, 1:800). The α-bungarotoxin (α-BTx) (cat. B35451, 1:200) and Alexa-conjugated secondary antibodies (1:200) were from Life Technologies. Unless otherwise stated, all other reagents were from Sigma. NUCC-390 was synthesized as previously described in [20 (link)].
The M. nigrocinctus venom was a pool of more than 50 specimens collected in the central and Pacific regions of Costa Rica and kept at the Serpentarium of Instituto Clodomiro Picado (University of Costa Rica). Once obtained, the venom was freeze-dried and stored at −20 °C.

Total cell lysates were prepared using M-PER Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Cat#78,503). A bicinchoninic acid protein assay (ThermoFisher Scientific) was used to estimate protein concentration. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a NuPAGE electrophoresis system, protein extracts were transferred to immobilon polyvinylidene difluoride membranes. The membranes were blocked in 5% milk and then incubated with anti-CXCL12 (Proteintech), anti-CXCR4 (Abcam), anti-TSPAN4 (Biossusa), anti-integrin β1 (Abcam), anti-actin (Abcam), anti-RhoA, and anti-PPAR-γ (Abcam) primary antibodies following the manufacturers’ instructions. Thereafter, the membranes were incubated with secondary antibodies and a WesternBreeze Chemiluminescent Detection Kit (ThermoFisher Scientific) was used to detect signals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control.

Anti-TSPAN4 (Biossusa, Cat#bs-9413R); anti-TSPAN7 (Biorbyt, Cat#orb373388); anti-integrin β1 (SantaCruz, Cat#sc374429); anti-CXCL12 (Proteintech, Cat#174021AP); anti-CXCR4 (Abcam, Cat#ab124824); anti-CD34 (Abcam, Cat#ab81289); anti-Perilipin (Progen, Cat#Gp40); anti-RhoA (Abcam, Cat#ab86297); anti-PPARγ (Abcam, Cat# ab209350) antibodies; goat anti-rabbit Alexa Fluor® 488 IgG (Abcam, Cat# ab150077); goat anti-guinea pig Alexa Fluor® 647 IgG (Abcam, Cat# ab150187); goat anti-rabbit Alexa Fluor® 647 IgG (Abcam, Cat#ab150079); goat anti-mouse Alexa Fluor® 488 IgG (Abcam, Cat#ab150113); AMD3100 (Abmole, Cat#M1898); CCG-1423 (Abmole, Cat#M8999); culture medium for mouse ASCs (OriCell, Cat#MUXMD9011); PBS (Gibco, Cat#10,010,023); trypsin–EDTA (Gibco, Cat#25,200,056); siRNA targeting CXCL12 (SANTA CRUZ, Cat#sc-39,368).

Exosome-derived proteins were extracted from exosomes using a Qproteome Mammalian Protein Prep Kit (Qiagen). Proteins from cell lysates were then analyzed using western blotting. Cells were first harvested and then suspended in RIPA lysis buffer (Beyotime, Haimen, China) containing 1 mM PMSF and a protease inhibitor cocktail. Protein content was quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, United States) and then separated with 10% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, United States), blocked with 5% BSA, and then incubated overnight at 4°C with the following primary antibodies: anti-hnRNPA2B1 (1:1,000; Abcam), anti-CXCL12 (1:1,000; Bioss, Beijing, China), anti-CXCR4 (1:100, Abcam) and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) for 1 h at room temperature. Enhanced chemiluminescent (ECL) reagent (Pierce, Rockford, IL, United States) was used to visualize the signals.

The total protein lysate of cells was prepared in radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). A bicinchoninic acid protein assay kit (Beyotime) was used to determine the protein concentrations. Equal amounts (20 μg) of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Subsequently, the membrane was incubated in 5% nonfat milk in Tris-buffered saline (TBS) for 1 h at room temperature. The following primary antibodies were incubated with the membrane at 4°C overnight: anti-CXCR4 (cat:#ab181020, 1 : 1,000) from Abcam (Cambridge, MA, USA), anti-AFP (cat:#4448, 1 : 1,000), anti-FOXM1 (cat:#20459, 1 : 1,000), phosphor-FOXM1 (cat:#14170, 1 : 1,000), and anti-p65 (cat:#3033, 1 : 1,000) from Cell Signaling Technology (Danvers, MA, USA), as well as anti-β-actin (cat:#66009-1-Ig, 1 : 5,000) from Proteintech. After washing with 0.5% Tween-20 in TBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Absin, Shanghai, China) for 1 h at room temperature. Protein signals were developed with enhanced chemiluminescence.

The following primary antibodies were used at the indicated dilutions: anti-CXCR4 (Abcam, cat. Ab 124824, 1:400), anti-syntaxin (Synaptic System, cat. 110111.00, 1:200), anti-Neurofilament (Abcam, cat. Ab 4680, 1:800). α-bungarotoxin (α-BTx) (cat. B35451, 1:200) and secondary antibodies Alexa-conjugated (1:200) were from Life Technologies. Unless otherwise stated, all other reagents were from Sigma. NUCC-390 was synthesized as previously described [46 (link)]
Purified Taipoxin and Oxyuranus scutellatus total venom were from Venom Supplies (Tanunda, South Australia). We estimated that our batch of taipoxin has a mouse LD50 of about 2 μg/Kg and that the Taipan venom is about 8 μg/Kg closely similar to value reported in the literature [47 (link)].