Anti gm130

Each sample were electrophoresed on SDS-PAGE gels and were transferred to nitrocellulose membranes. The membranes were probed with specific antibodies as follows; anti-CD9 (Abcam, Cambridge, MA, USA) and anti-GM-130 (Abcam). The membranes were incubated with horseradish peroxidase-coupled secondary antibody (Sigma). Following washing with TBS-T, the bound antibody was detected by enhanced chemiluminescence (Amersham, Buckinghamshire, UK).

Milk-derived exosomes for western blotting were suspended in 100 μl RIPA buffer (Solarbio, Shanghai, China). The exosomal surface proteins were analyzed by western blotting as described previously.^{10 (link)} Blots were probed for CD9, CD63, Tsg101 (Abcam, Cambridge, UK) using secondary antibody anti-GM130 (Abcam, Cambridge, UK). All antibodies were used following the manufacturer's instructions.

SCs were plated onto 35 mm diameter dishes in complete medium (DMEM + 10% FBS + 2 µM forskolin). Cells were washed twice with PBS and fixed for 20 min in 4% paraformaldehyde (PFA, Sigma-Aldrich, Milan, Italy) in PBS, at RT. After three washes in PBS, SCs were incubated for 45 min in 0.1% Triton X-100 (Sigma-Aldrich, Milan, Italy), 10% normal goat serum (NGS, Vector Laboratories, Burlingame, CA, USA) and 1% bovine serum albumin (BSA; Sigma-Aldrich, Milan, Italy) in PBS. Then, the cells were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Neuregulin-1 (H210)(1:200, Santa Cruz, sc-28916, Dallas, TX, USA); rabbit polyclonal anti-HER2/ErbB2 (C18), (1:300, Santa Cruz sc-284, Dallas, TX, USA); mouse polyclonal anti-GM130, (1:300, Abcam, ab169276, Cambridge, UK); mouse monoclonal anti-Lamp1 (H4A3), (1:200, Abcam, ab25630, Cambridge, UK); rabbit polyclonal anti-GRP78 BIP, (1:300, Abcam, ab53068, Cambridge, UK), diluted in 0.1% Triton X-100, 1% NGS, 1% BSA in PBS. After three washes in PBS, SCs were incubated for 1 h at RT with the appropriate secondary antibodies: goat anti-rabbit or mouse IgG- Alexa 488 or 594-conjugated (Promega, Milan, Italy), diluted 1:500 in PBS + 0.1% Triton X–100 + 1% NGS. After three washes in PBS, the slides were mounted with Vectashield (H1200, Vector Lab, DBA, Milan, Italy).

We used the following antibodies: anti-TOMM20 (Abcam, USA, cat. ab56783); anti-GM-130 (Abcam, USA, cat. ab52649); anti KDEL (Abcam, USA, cat. ab12223). Control and treated macrophages seeded on chamber slides (0.4 × 10⁶/slide) were fixed in 4% formaldehyde in PBS with 0.05% Triton X-100. After washing in PBS–Tween 20, fixed cells were blocked in casein blocking buffer (Bio-Rad, USA) with 0.05% Tween 20 and subsequently incubated in blocking buffer with a 1:200 dilution of primary antibodies and rhodamine–phalloidin overnight at 4 °C. Subsequently, after extensive washing in PBS–Tween 20, cells were mounted in ProLong^® Diamond Antifade with DAPI and observed under a Nikon fluorescence microscope. All experiments were performed in triplicate. The difference in organelle distribution between untreated and treated cells was calculated using the fluorescent area (normalized to total cell area using actin fluorescence) and corrected total cell fluorescence of individual cells using the image-processing ImageJ program.

24–48 h post-transfection, confluent cells were washed twice with PBS⁺⁺ (pH 8, 1 mM MgCl₂, and 0.1 mM CaCl₂). Cells were then fixed with 2% paraformaldehyde in PBS for 20 min at room temperature, incubated with 50 mM NH₄Cl, permeabilized with 0.1% Triton X-100 for 1 min and incubated with DAKO (antibody diluent with background-reducing components) for 30 min to block no specific antibody binding. Fixed cells were incubated for 1 h at room temperature with the primary antibodies at appropriate dilution in DAKO. Mouse anti-Myc was visualized with Texas Red-coupled or Alexa Fluor 647 conjugated secondary antibodies (Thermo Fisher Scientific, Paris, France). Rabbit anti-calnexin (Abcam, Paris, France) was visualized with FITC-coupled antibodies (Themo Fisher Scientifc). Anti-Giantin (Abcam), anti-GM130 (Abcam), and LAMP2 (Abcam) were with Alexa Fluor 555 conjugated secondary antibodies (Thermo Fisher Scientific). Cells were then washed with PBS and mounted with Vectashield.

Total proteins from cells or exosomes were extracted at 4 °C for 30 min by using RIPA Lysis Buffer (Beyotime, China). Adipose tissue samples were homogenized using a Teflon/glass homogenizer in lysis buffer with pH 7.4, containing 50 mM Tris-HCL buffer, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 50 mM NaF, 1 mM orthovanadate, and protease inhibitors. Then, the lysates were centrifuged at 500g for 10 min at 4 °C and the supernatant was collected. Western blotting was performed according to the concentration of proteins determined by BCA method. Primary antibodies used in this study included anti-GM130 (1:1000, Abcam, ab30637), anti-CD63 (1:1000, Abcam, ab134045), anti-TSG101 (1:500, Santa, sc-7964), anti-CD9 (1:1000, Abcam, ab236630), anti-GAPDH (Proteintech, 60004-1-1 g) and anti-BMP7 (1:1000, Abcam, ab129156). Secondary antibodies were HRP-conjugated goat anti-mouse IgG (D110087, BBI, China) and HRP-conjugated goat anti-rabbit IgG (1:5000, Cell Signaling Technology, 7074P2).