Ripa cell lysate

After 48 h of EEC treatment, the original culture medium was discarded and cells were washed with PBS three times, placed on ice, and mixed with RIPA cell lysate (Applygen Technologies Inc., Beijing, China), containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The protein extraction method and WB analysis have been described previously [9 (link),23 (link)]. All test antibodies are shown in Table 2. The blots were visualized using enhanced chemiluminescence (Advansta, Menlo Park, CA, USA), and related data were analyzed using ImageJ processing software; each experiment was performed in triplicate.

We cut 50 mg of sample and put it into a centrifuge tube. Then, it was added 1 mL
of RIPA cell lysate (C1053; APPLYGEN). Afterwards, we ground it thoroughly in a
grinder (65HZ, 60s) to a homogenate; centrifuged it at 12.000 rpm, lasting 15
min; and carefully aspirated the supernatant, to obtain the total protein. We
assayed the protein concentration as per BCA Protein Assay Kit (CW0014S; CWBIO).
Subsequently, the protein was denatured, loaded, and subjected to SDS-PAGE,
lasting 2 h. Then, it was transferred to the PVDF Membrane (IPVH00010;
Millipore), with a constant current of 300 mA, lasting 80 min. We incubated the
membrane with primary antibodies—anti-TGF-β1 (bs-0068R, Bioss, 1/500),
anti-Smad2 (AF6449, Affinity, 1/1000), anti-Smad3 (ab40854, Abcam, 1/1000), and
anti-GAPDH (TA-08, ZSGB-Bio, 1/2000)—overnight at 4 °C; then with HRP-labeled
secondary antibodies—HRP-labeled goat anti-mouse IgG (H+L) (ZB-2305, ZSGB-Bio,
1/2000), HRP-labeled goat anti-rabbit IgG (H+L) (ZB-2301, ZSGB-Bio, 1/2000)—,
lasting 2 h, at indoor temperature. ECL luminescent liquid (RJ239676; Thermo
Fisher Scientific) was dropped onto the membrane and exposed in a gel imaging
system. Ultimately, we used Quantityone software for analyzing the gray value of
each antibody band.