Anti mmp11

Cells were lysed in RIPA buffer with protease and phosphatase inhibitors (PMSF 1 mM, aprotinin 10 μg/mL, leupeptin 10 μg/mL, Na₃VO₄ 1 mM and NaF 20 mM). Protein concentration was measured by the BCA method (#10753505, #10341664, #10495944; Thermo Scientific), using a standard curve prepared with known concentrations of bovine serum albumin (BSA). Samples were sonicated for 2 minutes (20 seconds on and 20 seconds off cycles) and prepared in Laemmli buffer, followed by a 5‐minute incubation at 95°C. Equal concentration of protein was adjusted with RIPA buffer. SDS‐PAGE gels were transferred to PVDF membranes, blocked with 5% non‐fat milk in tris‐buffered saline 0.1% Tween (TBST) solution for 1 hour and then incubated with the specific primary antibodies (1:1000 dilution) (anti‐FLAG: F3785, Sigma; anti‐β‐actin: #3700, CST; anti‐MMP11: SN74‐08, Invitrogen; anti‐COL1A1: #72026, CST; anti‐COL4A1: 55131‐1‐AP, Proteintech). Membranes were washed with TBST and incubated with anti‐mouse (sc‐2005, SCB) or anti‐rabbit (sc‐2004, SCB) HRP‐conjugated secondary antibodies in 5% BSA TBST for 1 hour (1:5000 dilution). HRP luminescence was stimulated using the Clarity Western ECL Substrate (#170‐5061, Bio‐Rad) and detected with a VWR Chemi Imager Premium station. ImageJ was used for quantification.

Western blotting was performed as previously reported.⁵¹ (link) Antibodies used in the experiments were anti-CD9 (CST, #13403), anti-TSG101 (Abcam, #ab125011), anti-ZEB1 (CST, #3396), anti-HIF1α (CST, #36169), anti-E-cadherin (CST, #3195), anti-vimentin (CST, #5741), anti-GLUT4 (CST, #2213), anti-PPARγ (CST, #2435), anti-HSL (CST, #18381), anti-α-SMA (CST, #19245), anti-MMP11 (Invitrogen, #SN74-08), anti-collagen I (CST, #72026), and anti-GAPDH (CST, #5174). The mouse IL-1β in culture medium was measured in the coculture or non-coculture experimental conditions using a Mouse IL-1β ELISA Kit (Dakewe Biotech, #2012096).