Bca protein assay kit

Protein extraction and Western blotting were performed following the protocol that we previously reported [23 (link), 24 (link)]. The cultured cells or the mouse lung tissues were homogenized in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (P1045, Beyotime, Shanghai, China) on ice for 30 min. The lysates were then centrifuged at 18,000g for 10 min at 4 °C, and the supernatant was collected and stored at −80 °C until needed. Soluble protein concentrations were determined using the BCA protein assay kit (PH0326, Phygene, China) following the manual’s instructions. Protein lysates (20–50 µg) were denatured and separated on a 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). This was followed by membrane blocking with Tris-buffered saline/0.05% Tween-20 (TBST) containing 5% w/v skim milk for 1 h at room temperature. After washing with TBST three times for 10 min each, the membranes were incubated at 4 °C overnight with the primary antibodies against YAP (1:1000 diluted, sc-101199, Santa Cruz), phosphor-S127 YAP (1:1000 diluted, 13,008, Cell Signaling Technology), TAZ (1:1000 diluted, ab224239, Abcam), non-pS127 YAP (1:1000 diluted, ab205270, Abcam), vimentin (1:1000 diluted, 5741, Cell signaling), α-SMA (1:1000 diluted, A2547, Sigma), CTGF (1:1000 diluted, ab6992, Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:5000 diluted, 2118S, Cell signaling Technology), or β-actin (1:100,000 diluted, AC026, ABclonal, USA). Subsequently, the membranes were incubated for 1 h with the secondary horseradish peroxidase (HRP)-conjugated IgG (1:3000 diluted for HRP-conjugated goat anti-rabbit IgG, 7074S, Cell signaling; 1:5000 diluted for HRP-conjugated goat anti-mouse IgG, 7076S, Cell Signaling Technology). After membrane washing with TBST, enhanced chemiluminescence (ECL) was conducted using the GenSuper ECL kit (JXE0011, Jingxin, China), and the protein bands were visualized on the iBright FL1500 system (Thermofisher, CN, USA). Densitometry was performed using ImageJ software (1.53c, Wayne Rasband, National Institutes of Health, USA), and GAPDH or β-actin were used to normalize the equal sample loading.

Total protein was extracted from cells using RIPA Lysis Buffer (Phygene, China) and quantified using the BCA Protein Assay Kit (Phygene). Proteins were separated using 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against LRRC15 (1:1,000, ab150376; Abcam, UK), RUNX1 (1:1,000, PA5-85543; Thermo Fisher Scientific, USA), CBF-β (1:1,000, ab231345; Abcam), and GAPDH (1:2,000, #5174; Cell Signaling Technologies, USA). After that, the membranes were further incubated with horseradish peroxidase (HRP)-coupled secondary IgG antibody (1:10,000, ab6721; Abcam) for 120 min at room temperature. Protein bands were examined using an ECL substrate kit (Abcam), and all proteins were quantified based on the internal reference band GAPDH using ImageJ software.