Anti cd63 sc 5275

VE-cadherin was detected using a mouse monoclonal antibody (sc-9989, Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 1:100 dilution for immunofluorescence and at 1:2000 dilution for immunoblotting. Connexin43 (Cx43) was detected using rabbit polyclonal antibodies directed against amino acids 363–382 of human/rat Cx43 (C6219, SIGMA-Aldrich, Saint Louis, MO, USA) at 1:250 dilution for immunofluorescence and at 1:5000 dilution for immunoblotting. Immunoblotting for EV markers was performed using primary mouse monoclonal antibodies anti-flotillin-1 (sc-133153, Santa Cruz, CA, USA) diluted 1:200 and anti-CD63 (sc-5275, Santa Cruz, CA, USA) diluted 1:500. The secondary antibodies, AlexaFluor 488 goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG antibodies, were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Immunoblotting was performed as described earlier [32 (link),33 (link)].

MSC-exos, cells and kidney tissues were lysed in RIPA lysis buffer (ThermoFisher) and the protein concentration was detected using BCA assay (ThermoFisher). Protein samples were subjected to Bis-Tris Gel (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 10 min and incubated overnight with primary antibodies as follows: anti-CD9 (ab92726, Abcam), anti-CD63 (sc-5275, Santa Cruz), anti-Tsg101 (sc-7964, Santa Cruz) anti-Alix (sc-53540, Santa Cruz), anti-GM130 (12480, Cell Signaling Technology), anti-CD44 (ab189524, Abcam), anti-ICAM-1 (MA5407, Invitrogen), anti-VCAM-1 (39036, Cell Signaling Technology), anti-LFA-1 (ab13219, Abcam), anti-integrin α4 (8440, Cell Signaling Technology), anti-integrin β1 (sc-374429, Santa Cruz), anti-PCNA (sc-56, Santa Cruz), anti-CDK1 (sc-54, Santa Cruz), anti-caspase-3 (ab13847, Abcam), anti-Cyclin B1(4138, Cell Signaling Technology), anti-p53 (2524, Cell Signaling Technology), anti-Bcl-2 (sc-7382, Santa Cruz), and anti-Bax (sc-20067, Santa Cruz). Secondary antibodies were used for detection by an ECL advanced system (GE Healthcare). Intensity values expressed as the relative protein expression were normalized to β-actin (AB2001, Abways) or GAPDH (AB2000, Abways).

HAuCl₄, sodium citrate dihydrate, APTES, BSA, 3‐mercaptopropionic acid (MPA), N‐hydroxysuccinimide, penicillin, and streptomycin were purchased from Sigma‐Aldrich (St. Louis, MO). 1‐ethyl‐3‐(3‐(dimethylamino)propyl)carbodiimide (EDC) was purchased from Daejung Chemicals (KR). Anti‐CD9 (sc‐13118), anti‐CD63 (sc‐5275), and anti‐EGFR (sc‐373746) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). RPMI 1640 and fetal bovine serum (FBS) were purchased from GE Healthcare (Chicago, IL). A cell lysis buffer (10×) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Abcam (UK). EGFR lyophilized powder (E2645) for the off‐target test and imaging was purchased from Sigma‐Aldrich (St. Louis, MO). GNP colloidal solutions of one optical density were purchased from nanoComposix (San Diego, CA). Full‐length EGFR ELISA kit was purchased from Invitrogen (Carlsbad, CA)

The quantity of protein in the EV samples was calculated using BCA assay (Pierce™ BCA Protein Assay Kit) according to the manufacturer’s recommendations. Equal amounts of total protein (10 µg) were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (741,280, BioTop, Germany). The membranes were then incubated overnight at 4 °C with the following primary antibodies (anti-HSP70 [sc-373867], and anti-CD63 [sc-5275]) from Santa Cruz Biotechnology and then with the secondary antibody (P0260, Aligent Tech, Glostrup, Denmark). The blots were then developed using Pierce™ ECL plus Western blotting substrate (32,132, Thermo Fisher Scientific, United States).

In order to confirm the presence of EVs, the EV surface markers CD63 [70 (link)] and CD81 [71 (link)] were analyzed by Western blot. For this purpose, the EV-containing elution fraction (see Section 4.5) was concentrated using Amicon^® ultra filters 2 mL 3 kDa (Merck, Darmstadt, Germany). Concentrates were added with RIPA lysis buffer supplemented with pefabloc (1 mg/mL), aprotinin (1 µg/mL), pepstatin A (1 µg/mL), and leupeptin (5 µg/mL) as protease inhibitors. To compare the expression of ABCC2 and ABCG2 in different cell types, cellular proteins were purified as described in 4.4. Proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Subsequently, membranes were blocked with 5% bovine serum albumin for at least 1 h and incubated overnight with the primary antibodies anti-CD63 (sc-5275, 1:200 in blocking buffer), anti-ABCG2 (BXP21, 1:100 in blocking buffer) from Santa Cruz Biotechnology (Heidelberg, Germany), anti-CD81 (555675, 1:200 in blocking buffer) from BD Biosciences (Heidelberg, Germany), or anti-ABCC2 (M2-III-6, 1:100 in blocking buffer, Enzo Life Sciences, Farmingdale, USA). Mouse IgG HRP-Linked Whole Antibody (1:2000, Cytiva, Marlborough, MA, USA) was used as the secondary antibody. Immunoreactive bands were detected by chemiluminescence using the ChemoStar touch device (Intas Science Imaging Instruments GmbH, Göttingen, Germany).