Anti tsg101

Homo sapiens HepG2 (ATCC HB-8065) and Hela (ATCC CCL-2) cells were cultured as a monolayer at 37 °C in complete Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco, USA) under a humidified atmosphere containing 5% CO₂. Fathead minnow (FHM) cells (ATCC CCL-42) were grown in M199 medium supplemented with (Gibco, USA) 10% (vol/vol) FBS at 27 °C.
Rabbit and mouse polyclonal antisera against TFV MCP were previously generated in our laboratory. Rabbit monoclonal antibody against the MYC tag and anti-Nedd4 antibody was purchased from Abcam (UK). Rabbit monoclonal anti-GAPDH antibody was obtained from CST (BRD). Mouse monoclonal anti-Tsg101, anti-Alix, anti-VPS4B, and rabbit polyclonal anti-VPS4 antibody were acquired from Santa Cruz (USA). Anti-rabbit/mouse IgG (H+L)-HRP conjugates were purchased from Promega (USA). All antibodies were used according to the manufacturers’ instructions.

The expression of protein was assessed by western blot analysis, and its expression in the samples was normalized to β-actin expression. Cells and tissues were lysed in RIPA buffer with freshly added protease inhibitor cocktail. Total lysates were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore). The immunoblots were blocked with 5% BSA at room temperature for 1 h and incubated at 4 °C overnight with anti-HIF-1α (1:200, Santa Cruz), anti-GEF-H1 (1:1000, Cell Signaling), anti-RhoA (1:500, Immunoway), anti-E-cadherin (1:500, Santa Cruz), anti- β-actin (1:3000, Santa Cruz), anti-CD63 (1:2000, Abcam), and anti-TSG101 (1:1000, Santa Cruz). After incubation with the secondary antibody, the membranes were visualized with an enhanced chemiluminescence system kit (Millipore, USA) according to the manufacturer's protocol.

ExoE-EVs (10uL) were deposited on Formvar carbon coated, glow-discharged grids using a Denton Desk III TSC Sputter Coater (Electron Microscopy Sciences, Cat. No. FF300-Cu). After 20 minutes of adsorption, grids were fixed with 4% PFA in NaCac for 10 minutes. Grids were then quenched with 50 mM of Glycine in PBS for five minutes then washed with PBS x 3 in 50 uL for 5 minutes each wash. The grids were incubated in blocking media containing 1% BSA in PBS for 20 minutes. After washing with PBS as indicated above, EVs were exposed to either anti-CD63 (LifeTechnologies, Cat. No. 10628D) or anti-Tsg101 (Santa Cruz, sc-7964) for 45 minutes followed by incubation with Protein A– 10nm Gold Conjugate for 40 minutes at room temperature (Cytodiagnostics, DKU: AC-10-05). Grids were fixed in 1% Glutaraldehyde and 2% PFA in NaCac. The grids were then stained with 4% uranylacetate in 0.15M Oxalic Acid, pH 7, embedded with 2% methylcellulose in 4% uranylacetate, and imaged in a JEOL, JEM1400 is a 120kV Transmission Electron Microscope with a LaB6 emitter and imaged using a Gatan Orius 10.7 megapixel CCD camera. Images were taken at either 25,000X or 40,000X.

Proteins were extracted from exosomes or cells using RIPA lysis buffer, and immunoblotting was performed as previously described [15 (link)]. Anti-CD63, anti-TSG101, anti-calnexin, anti-hnRNPA1, and anti-GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-HEYL was from Abcam (Cambridge, UK).

The HGF, cMET and EGFR expression was assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at RT for 1 h and incubated at 4 °C overnight with anti-HGF (1:1,000, Abcam), anti-EGFR (1:5,000, Abcam), anti-cMET (1:1,000, Abcam), anti-p-cMET (1:1,000, Abcam), anti-CD63 (1:2,000, Abcam), anti-TSG101 (1:1,000, Santa Cruz), anti-Alix (1: 1,000, Santa Cruz), anti-F4/80 (1:1,000, Abcam), anti-desmin (1:1,000, Immunoway), anti-α-SMA (1:1,000, Santa Cruz) and anti-GAPDH (1:3,000, Santa Cruz) antibodies, respectively.

The SDF-1α, Arx, Pax4, Insulin and Glucagon expression were assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated at 4°C overnight with anti-SDF-1α (1:1000, Santa cruz), anti-Arx (1:1000, Santa cruz), anti-Pax4 (1:1000, Santa Cruz), anti-Insulin (1:1000, Santa cruz), anti-Glucagon (1:1000, Santa cruz), anti-Aldh1a3 (1:1000, Novus), anti-Neurog3 (1:1000, Beta Cell Biology Consortium), anti-MafA (1:1000, Cell Signaling Technology), anti-Pdx1 (1:1000, Cell Signaling Technology), anti-NeuroD1 (1:1000, Cell Signaling Technology) and anti-XBP1 (1:1000, Santa cruz), anti-CD63 (1:2000, Abcam), anti-TSG101 (1:1000, Santa Cruz), anti-Ago2 (1: 1000, Santa Cruz) and anti-GAPDH (1:3000, Santa Cruz) antibodies respectively.

The uEVs were resuspended and diluted in a buffer that comprised 0.5% SDS and 1.1% Triton X-100. The process of size separation was undertaken through SDS PAGE gel electrophoresis. Afterward, the proteins were transferred onto polyvinylidene difluoride membranes and probed with specific antibodies, including anti-ALIX (Santa Cruz sc-53540), anti-TSG101 (Santa Cruz sc-7964), and anti-CD63 (Santa Cruz sc-5275). The testing was enhanced by verifying the samples using an anti-AQP2 (phospho S256) antibody (abcam 111346) and an anti-GSK3β (phosphor Y216) antibody (abcam 4797). Enhanced chemiluminescence facilitated the visualization of these tests.