Bca reagent

To perform the immunoblotting assay, all proteins were extracted using RIPA buffer supplemented with phosphatases and protease inhibitors (Bimake, #B15003 and #B14002). Proteins were quantified by BCA reagent (Yeasen, #20201ES90), separated by SDS-PAGE gel, and then transferred onto PVDF membranes. After blocking in 5 % Bovine Serum Albumin (BSA) (Proliant Biological, #69100) for 1 h, the membranes were incubated with indicated antibodies, including BTBD7 (Invitrogen, # PA5-45934), ROCK2 (CST, #47012), MMP-2 (CST, #40994), MMP-9 (CST, #13667), and GAPDH (CST, #5174) at 4 °C overnight. Following three washes in PBS, the membrane was treated with secondary antibodies. Finally, the signals were detected using an enhanced chemiluminescent (ECL) substrate kit (Yeasen, #36208ES80).

For immunoblotting assays, all proteins were extracted using RIPA buffer and quantified using BCA reagent (#20201ES90, Yeasen). Proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (#IPVH00010, Millipore). After blocking in 5% bovine serum albumin (BSA, #36101ES80, Yeasen) and incubating with specific antibodies, the bands were visualized using the enhanced chemiluminescence (ECL) detection kit (#36208ES76, Yeasen), and analyzed by ImageJ software. All antibodies used in this study are provided in Supplementary Table S6.

Tissue/Cells were lysed in RIPA Lysis Buffer (Beyotime, China) supplemented with PMSF (Beyotime, China) and protease inhibitors and phosphatase inhibitors (Sigma, USA). Nuclear or membrane extracts were prepared with a nuclear and cytoplasmic protein extraction kit (Beyotime, China) according to the manufacturer’s instructions. Protein concentrations were measured with the BCA reagent (YEASEN, China) by using a Microplate Reader Spark System (TECAN, Switzerland). Equal amounts of protein were resolved by SDS–PAGE and immunoblotted with different antibodies as described previously. The immunoblots were detected using an Image 600 ECL System (GE, USA).