Gar0072

For phenotypic characterization, MSCs and MSC-EVs were lysed via lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM Na₃VO₄, and 1 mM PMSF). Equal amount of total proteins from samples (20 μg or 40 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and detected via Western blot analysis. The primary antibodies used in the study included the following: CD63 (0.5 μg/ml; ab193349, Abcam, Cambridge, MA), CD81 (1:1000; sc-166029, Santa Cruz Biotechnology, Santa Cruz, CA), CD105 (1:1000; ab169545, Abcam), CD44 (1:5000; ab157107, Abcam), GM130 (1:5000; ab52649, Abcam), and Calnexin (1:5000; ab133615, Abcam). After washing, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase. Goat anti-Rabbit lgG (H+L) HRP Conjugated (1:5000, GAR0072, Multi Sciences, Hangzhou, China) and goat anti-Mouse lgG (H+L) HRP Conjugated (1:5000, GAM0072, Multi Sciences, Hangzhou, China) were used as Secondary antibodies. The signals were detected via enzyme-linked chemiluminescence using the EZ-ECL kit (Biological Industries, Kibbutz Beit-Haemek, Israel).

Harvested cells were lysed using M‐PER^TM Mammalian Protein Extraction Reagent (Thermo Scientific) with protease and phosphatase inhibitors (Beyotime). Protein concentration was determined by BCA protein quantification kit (Beyotime). Equal amounts of the proteins were loaded into SurePAGEᵀᴹ precast polyacrylamide gels with a gradient between 4% and 20% (GenScript) and transferred to PVDF membranes (Millipore) by eBlot^® L1 wet protein transfer system (GenScript). After blocking, the membranes were incubated with primary antibodies overnight at 4℃. Then, the membranes were incubated with HRP‐conjugated anti‐mouse or anti‐rabbit secondary antibody (GAM0072 or GAR0072, Multisciences). The protein bands were visualized by High‐sig ECL Western Blotting Substrate (Tanon). Images were collected using the Tanon‐5200 Chemiluminescent Imaging System (Tanon). The expression of β‐actin was detected as a loading control.
All primary antibodies were commercial products. Anti‐TAB182, anti‐CDC2 (sc‐137035), anti‐phospho‐CDC2 (sc‐136014) were purchased from Santa Cruz. Anti‐FHL2 was purchased from Proteintech. Anti‐CHK2 (ab207446), anti‐phospho‐CHK2 (ab32148) were purchased from Abcam; anti‐CDC25C (YM0142), anti‐phospho‐CDC25C (YP0058) were purchased from ImmunoWay. Anti‐β‐actin (ab008) was purchased from Multisciences.

Samples were homogenized in liquid nitrogen and thawed in RIPA buffer containing protease inhibitors. The protein abundances of GUCY1A3 (guanylate cyclase 1 soluble subunit alpha), GUCY1B3 (guanylate cyclase 1 soluble subunit beta), and PRKG1 (protein kinase cGMP-dependent 1) were normalized to GAPDH. The primary antibodies for GUCY1A3 (Proteintech, Cat#12605-1-AP), GUCY1B3 (Proteintech, Cat#19011-1-AP), and PRKG1 (Proteintech, Cat#21646-1-AP) were incubated overnight at 4°C. Secondary antibodies (goat anti-mouse for GAPDH, GAM0072; goat anti-rabbit for the others, GAR0072, Multisciences) were incubated for 1 h at room temperature. The immunoreactive bands were visualized using the UVP imaging system. Imaging signals were digitized and analyzed, and the ratio of band intensity to GAPDH was subsequently obtained to quantify relative protein expression.