Pvdf membrane

At the experimental endpoint, the eWAT from WT and siglec-E KO mice fed on ND or HFD was collected and quickly frozen in liquid nitrogen before further processing. Tissue lysates were prepared using RIPA buffer (Cat. no. J63306, Alfa Aesar, Haverhill, MA) with protease and phosphatase inhibitors. The resulting lysates were subjected to centrifugation (16000 x g for 20 min) to remove cell debris, the supernatant was collected and the protein concentration in each lysate was measured using a BCA protein assay kit (Cat. no. 23225, Thermo Fisher Scientific, Waltham, MA). An equal amount of protein in each lysate was mixed with 4X Laemmli sample buffer (Cat. no. 1610747, Bio-Rad, Hercules, CA) containing fresh 2-β-mercaptoethanol (Cat. no. A15890, Alfa Aesar, Haverhill, MA), and heated at 95°C for 5 min. The protein samples were subsequently separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Bio-Rad, Hercules, CA). After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Cat. no. 1620174, Bio-Rad, Hercules, CA) by using a Trans-Blot Turbo instrument (Bio-Rad, Hercules, CA). Membranes were blocked with intercept blocking buffer (cat. no. 927-60001, LI-COR Biosciences, Lincoln, NE) at room temperature for 1-2h and incubated with antibodies specific for TRAF3 (monoclonal Ab, cat. no. 66310-1-Ig, Proteintech), Akt2 (clone 8B7, cat. no. SC-81436, Santa Cruz Biotechnology), or Akt1/2/3 (clone BDI111, cat. no. SC-56878, Santa Cruz Biotechnology) overnight at 4°C with continuous shaking (Table 4). Later, the membranes were washed three times (5-10 min. each) with TBS containing 0.2% Tween 20 (cat. no. 28360, Thermo Fisher Scientific, Waltham, MA). The membranes were then incubated at RT for 1 hr with the appropriate secondary antibodies (1:5,000), namely IRDye^® 800C W-labeled goat anti-mouse IgG (cat. no. 926-32210, LI-COR Biosciences, Lincoln, NE), IRDye^® 680RD-labeled goat anti-mouse IgG (cat. no. 925-68070, LI-COR Biosciences), or goat anti-rabbit IgG (cat. no. 926–32211, LI-COR Biosciences), then washed three times (5-10 min. each) with TBS containing 0.2% Tween 20 (cat. no. 28360, Thermo Scientific Fisher, Waltham, MA). Images were taken with an LI-COR Odyssey^® DLX imaging system (LI-COR Biosciences, Lincoln, NE) and densitometric analyses were performed using ImageJ software (NIH).

Total proteins were extracted from ovarian tissues using a dedicated total protein kit. Briefly, the ovarian tissues were homogenized in lysis solution (R0010, Solarbio, China) with grinding beads (YA3031, Solarbio, China) and centrifuged at 2000 rpm for 1 min. In addition, the total proteins from GCs and hUSC-Exo were extracted by solely employing a lysis buffer to disrupt both the cells and the exosomes, thoroughly vortaxing the mixture for 30 s and repeating this process five times. The protein concentrations were determined by the bicinchoninic acid method (BCA; PC0020, Solarbio, China). The proteins were run on 10% denaturing SDS-PAGE gels, then transferred to polyvinylidene fluoride (PVDF) membranes (BioRad, USA), which were incubated with primary antibodies at 4 °C overnight. The antibodies used in the study were as follows: anti-GAPDH (1:5000, D4C6R, mouse monoclonal, CST), anti-FSHR (1:1000, CL594-22,665 rabbit monoclonal, protein technology), anti-Bcl-2 (1:1000, D55G8, rabbit monoclonal, CST), anti-P-AKT (1:1000, D9E, rabbit monoclonal, CST), anti-AKT (1:1000, 9272, rabbit monoclonal, CST), anti-P-mTOR (1:1000, D9C2, rabbit monoclonal, CST), anti-mTOR (1:1000, 7C10, rabbit monoclonal, CST), anti-P-PI3K (1:1000, E3U1H, rabbit monoclonal, CST), anti-PI3K (1:1000, 19H8,rabbit monoclonal, CST), anti-CD81 (1:1000, D3N2D, rabbit monoclonal, CST), anti-TSG101 (1:1000, E6V1X rabbit monoclonal, CST), anti-CD63 (1:1000, E1W3T rabbit monoclonal, CST), and anti-CD9 (1:1000, D8O1A rabbit monoclonal, CST). After 16 h, the blots were detected using horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody (Invitrogen, USA) for 1 h at room temperature. The images were quantified using the Super Signal West Pico chemiluminescence detection system.

For western blotting, the harvested cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific Inc.) supplemented with a proteinase inhibitor (Thermo Fisher Scientific Inc.) on ice for 30 min. The lysed cells were centrifuged for 13,000 r/min at 4 °C. The supernatant was harvested and used to quantify protein concentration using protein broad-range assay kits following the manufacturer’s protocol (Thermo Fisher Scientific Inc.). For each group, 20 μg of protein was mixed with SDS-PAGE sample buffer (Biopeace, Dajeon, Chungnam, South Korea) and loaded into mini-protean TGX gels (Bio-Rad, Hercules, CA, USA). For the western blotting assay, the same volume of each medium was used without a quantification step. The loaded gel was run at 100 V for 80 min, and the proteins were transferred to a PVDF membrane (Bio-Rad) at 0.3A for 2 h. The transferred membrane was blocked with an appropriate blocking buffer (Biopeace) and incubated with an anti-myostatin antibody (R&D Systems, Minneapolis, MN, USA) or anti-pSmad3 antibody (Thermo Fisher Scientific Inc.) overnight at 4 °C. The next day, the membranes were washed and incubated with HRP peroxidase-conjugated secondary antibody (Gendepot) for 1 h at room temperature. Chemiluminescent bands were visualized using iBright 1500 (Thermo Fisher Scientific Inc.) after treatment with an enhancer-peroxide solution mixture (Biopeace).

The Bicinchoninic Acid protein assay (BCA; Beyotime) was employed to isolate and detect all of the protein in the cell lines or tissues. Typically, 10% SDS polyacrylamide gels from Solarbio were employed to perform protein isolation which then underwent transferring onto PVDF membranes from Bio-Rad. The membranes underwent 60 min of blockage at room temperature with a solution consisting of PBS and 5% nonfat milk. Subsequently, the membranes underwent overnight incubation with primary antibodies targeting ADAMTS12 (ab45041, Abcam), p-AKT (ab38449, Abcam), COL3A1 (ab184993, Abcam), FAK (ab40794, Abcam), p-FAK (ab81298, Abcam), p-PI3K (ab182651, Abcam), AKT (ab8933, Abcam), PI3K (ab302958, Abcam), GADPH (ab8245, Abcam) at 4 °C. After that, three washes with PBST were conducted to membranes which were then, at room temperature, exposed for 1 h to a horseradish peroxidase-conjugated secondary antibody. The enhanced chemiluminescence (ECL) technique was utilized to detect the signals subsequent to incubation with the secondary antibody.

To determine protein expression, mature 3T3-L1 adipocytes were harvested using a lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing phosphatase and protease inhibitors on ice. Briefly, the cell lysates were centrifuged at 13,000× g for 20 min at 4 °C, and the protein was quantified using the BSA assay. Proteins were separated using Mini-PROTEAN TGX Gels (Bio-Rad) and transferred onto PVDF membranes (Bio-Rad). The transferred proteins were blocked in EverBlot Blocking buffer (Bio-Rad), and the membranes were incubated with each primary antibody at 4 °C with shaking. After incubation, the membrane was incubated with secondary antibodies at RT for 2 h. The antibodies used are listed in Supplementary Table S2.

Total cellular proteins were separated through SDS-PAGE and transferred on a PVDF membrane (BioRad, Hercules, CA). The membrane was incubated with primary and secondary antibodies sequentially (Table S3). The protein-antibody complex was detected using an enhanced chemiluminescence (ECL) detection system. Image analysis was performed to quantify protein expression levels with GAPDH as an internal control. Representative images of three independent experiments are shown in the Figures.