Anti alix antibody

Western blotting was conducted according to a protocol previously described [43 (link)]. Briefly, the tissues were lysed with radioimmunoprecipitation assay buffer (Applygen, Beijing, China) for 30 min and then homogenized using an ultrasound treatment at 100 W for 3 min and centrifuged at 12,000 × g for 20 min. Protein concentrations were quantified using the bicinchoninic acid assay. The proteins were then denatured with sodium dodecyl sulfate (SDS) protein loading buffer (Applygen) and were separated by SDS-polyacrylamide gel electrophoresis (15 μg per well). Samples were transferred to membranes and immunoblotted with the following primary antibodies: anti-GAPDH antibody (1:2000, Abcam, Cambridge, UK), anti-GAP43 antibody (1:1000, Abcam), anti-LAMP-2 antibody (1:500, Abcam), anti-SNAP25 antibody (1:1000, Abcam), anti-Alix antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-TSG101 antibody (1:2000, ThermoFisher Scientific), anti-CD63 antibody (1:1000, ThermoFisher Scientific), and anti-APOB antibody (1:1000, ThermoFisher Scientific).

Samples were initially fixed with 4% paraformaldehyde for 10 min at room temperature. Then, samples were processed with 0.1% Triton X for 5 mins, followed by 5% Bovine serum albumin blocking for 1 h at room temperature. After staining with primary antibody overnight at 4 °C (Anti-Ascl1 antibodies, 1:100, Thermo Fisher Scientific, 14579482; anti-CD31 antibody, 1:100, BD Biosciences, 565629; anti-NeuN antibody, 1:200, Millipore, MAB377; anti-doublecortin antibody, 1:100, Cell Signaling Technology, 4604; anti-Nestin antibody, 1:100, Abcam, ab11306; anti-GFAP antibody, 1:200, Innovative Research, 13-0300; anti-MAP2 antibody, 1:100, Abcam, ab32454; anti-TUBB3 antibody, 1:100, BioLegend, 801209; anti-GFP antibody, 1:100, Santa Cruz Biotechnology, sc-9996; anti-Alix antibody, 1:100, Cell Signaling Technology, 2171; anti-PSA-NCAM, 1:100, Thermo Fisher Scientific, 14911882; anti-SOX2 antibody, 1:200, Millipore, AB5603; anti-PAX6 antibody, 1:200, Thermo Fisher Scientific, 13B10-1A10; anti-Ki67 antibody, 1:200, Abcam, ab15580). After washing off the primary antibody with PBS, the samples were then incubated with secondary antibodies (1:200; Jackson Immunoresearch Laboratories) for 1 h at room temperature. After that, the samples were covered with VECTASHIELD with DAPI (Vector Laboratories). Immunostaining was analyzed with a fluorescence microscope (Nikon).

Cargo proteins were extracted from EVs by heating the vesicles at 60 °C for 15 min in 100 mM TrisHCl buffer containing 4% SDS. Proteins released from the ruptured vesicles were then mixed with gel loading buffer and boiled at 95 °C for 10 min. Protein samples were separated by SDS-PAGE on 12% polyacrylamide gels and then transferred onto nitrocellulose membranes at 100 V for 1 h. The membranes were probed with primary antibodies at 4 °C overnight. The antibodies used for Western blots were: anti-Alix antibody (#2171), anti-flotillin-1 antibody (#18634), anti-CD9 antibody (#13174), anti-caveolin-1 antibody (#3267S) from Cell Signaling Technologies (Danvers, MA, USA). The anti-hepatocyte growth factor receptor antibody (ab59884) and anti-FTH1 (ab75973) antibodies were from Abcam (Cambridge, UK). Anti-E-cadherin (sc-21791), anti-N-cadherin (sc-59987), anti-cathepsin B (sc-365558), anti-cathepsin D (sc-377299) and anti-IGFBP3 (sc-9028) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PLOD2 (408105) antibody was obtained from Thermo Fisher Scientific. Protein–antibody conjugates were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific).

SK-N-SH cells were reverse transfected with 20 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) in collagen-coated 48-well plates and then cultured for 60 h. Knockdown efficacy was checked by immunoblotting (anti-Alix antibody [catalog no. 92880; Cell Signaling], anti-Nedd4 antibody [catalog no. 2740; Cell Signaling], anti-Nedd4-like antibody [catalog no. 4013; Cell Signaling], and anti-TSG101 antibody [catalog no. 28283-1-AP; Proteintech]) or qRT-PCR. siRNA-treated cells were infected with virus at an MOI of 1, and the supernatants were collected at 48 to 60 hpi.