Exposure meter

Total protein from MM cells was extracted with radioimmunoprecipitation assay (RIPA) buffer, and supernatants were collected for western blot assays. Proteins (20–40 μg) were separated on 8–12% pre-made protein electrophoresis gels and transferred to polyvinylidene difluoride membranes (Merck Millipore, Germany). The membranes were blocked with 5% nonfat milk or bovine serum albumin (BSA) for 1–2 h and then incubated with specific primary antibodies overnight at 4 °C. The membranes were washed for 15 min in Tris-buffered saline with Tween 20 (TBS-T) four times and then incubated with secondary antibodies at room temperature for 1 h. The membranes were washed with TBS-T, and bands were detected using an exposure meter (Bio-Rad, CA, USA) with an enhanced chemiluminescence detection kit for HRP (Biological Industries, Israel, Beit Haemek Ltd.).

We extracted the total protein by RIPA lysate. Then, we separated 30 μg protein on 10% protein electrophoresis gel and then transferred it to the PVDF membrane (Invitrogen). The PVDF membrane was blocked with 5% skim milk powder for 1-2 h, and then anti-collagen III (COLIII) (1 : 1000, Abcam, ab184993), anti-α smooth muscle actin (α-SMA) (1 : 500, Abcam, ab5694), anti- matrix metalloproteinase (MMP)-1 (1 : 1000, Abcam, ab52631), anti-MMP-8 (1 : 1000, Abcam, ab81286), anti-NFAT5 (1 : 1000, Abcam, ab3446), antitumor necrosis factor-α (TNF-α) (1 : 1000, Abcam, MA5-23720), interleukin (IL)-6 (1 : 500, Abcam, M620), anti-IL-1β (1 : 500, Abcam, M421B), and anti-β-actin (1 : 1000, Abcam, ab6276) were added and incubated overnight 4 °C. The next day, the PVDF membranes were incubated in HRP anti-mouse IgG for IP (1: 1000, Abcam, ab131368) at 25 °C for an h. The bands were detected using an exposure meter (Bio-Rad, USA) with hypersensitive chemiluminescence (HRP) detection kit (LMAI Bio, Shanghai, China).