Ab260033

Bone marrow mononuclear cells (BMMNCs) from healthy donors and leukemic blasts from AML patients were shifted to slides via cytospin, disposed with 3% H₂O₂ and blocked with 5% goat serum. The slides were then incubated at 4 °C overnight with primary antibodies against GPER (ab260033, 1:200, Abcam, Cambridge, UK), incubated using secondary antibody for 25 min, and stained with diaminobenzidine. Image-Pro 6.0 software was used to acquire and assess the photos (Media Cybernetics, Maryland, USA).

The total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease inhibitor and phosphatase inhibitors (Beyotime, Nanjing, China) and quantified by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Total protein extracts (30 µg) from exosomes, cells, and submandibular gland tissue were separated via 10% SDS‑PAGE (Epizyme, Shanghai, China) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) and blocked with 5% non‑fat milk for 1 h at room temperature. After incubation with the primary antibody and the secondary antibody, the target protein was visualized by chemiluminescence using an ECL kit (Beyotime, Nanjing, China). The antibodies used in the Western blot assay are listed as follows: GAPDH (1:10,000, ab181602, Abcam), AQP5 (1:1000, sc-514022, Santa Cruz), CD63 (1:1000, ab134045, Abcam), TSG101 (1:1000, ab125011, Abcam), ALIX (1:1000, ab275377, Abcam), GPER (1:1000, ab260033, Abcam), CREB (1:1000, ab32515, Abcam), p-CREB (1:5000, ab32096, Abcam), PKA (1:1000, #4782, Cell Signaling Technology), p-PKA (1:1000, #4781, Cell Signaling Technology) and goat anti-rabbit IgG H&L (HRP) antibody (1:5000, #31460, Thermo Fisher Scientific).

The LX-2 cells or liver tissues from CCl₄-induced or SSd treatment mice were lysed using RIPA Buffer (Solarbio, Beijing, China), and the total protein concentration was detected by BCA protein assay kit (Cell Signaling Technology, Danvers, MA, USA). The proteins were separated by 10% SDS-PAGE, and then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in TBST, the membranes were incubated with primary antibodies against p62 (1:2000, ab109012, Abcam), LC3 (2 µg/ml, ab128025, Abcam), GPER1 (1:1000, ab260033, Abcam), α-SMA (1:1000, ab108424, Abcam) and β-actin (1:5000, ab6276, Abcam) overnight at 4 °C. The blots were incubated with HRP-conjugated secondary anti-rabbit (1:5000) for 1 h at room temperature. Lastly, Immunoreactivities were visualized by chemiluminescence using the ECL kit (Pierce, Rockford, IL, USA). The intensity of protein bands on the Western blot image was quantified using ImageJ software.