Anti yap

LOXL2, YAP, and α-SMA protein expressions were analyzed using MSC-ex or BAPN-treated mouse tissue samples. Mouse tissue sections (4 µm thick) of formalin-fixed, paraffin-embedded liver specimens were deparaffinized in xylene and rehydrated in graded alcohol. Standard immunohistochemical procedures were performed on liver tissue sections using anti-LOXL2 (1:50; Santa Cruz Biotechnology, USA), anti-YAP (1:50; Bioworld, USA), or anti-α-SMA antibody (1:50; Bioworld, USA). Signals were visualized using 3, 3′-Diamino-benzidine tetrahydrochloride (Boster Biology, Wuhan, China). A brown membrane, cytoplasmic, and nuclear staining indicated a positive reaction according to different markers. The staining result of LOXL2, YAP, and α-SMA expression was determined by the percentage of positive cells by two investigators blinded to the data. Immunofluorescence staining was performed using anti-CD9 (1:100, Bioworld, USA), anti-LOXL2 (1:50), anti-YAP (1:50), or anti-α-SMA antibody (1:50). Negative controls with isotype IgG were run in parallel. Images were acquired using a laser scanning confocal microscope (Nikon, Tokyo, Japan).

Chromatin immunoprecipitation (ChIP) assay followed the manufacturer’s instructions (Millipore, Billerica, MA). Briefly, formaldehyde was used to cross-link proteins with DNA, and 2 × 10⁷ 293T or LX-2 cells were lysed in sodium dodecyl sulfate lysis buffer. The cell lysate was sonicated to shear the DNA to 400- to 600-bp lengths. Chromatin samples were then precleared with a salmon sperm DNA/protein A agarose 50% slurry for 30 min at 4 °C and immunoprecipitated overnight with anti-IgG or anti-YAP (Bioworld, USA, BS9920M). The purified DNA fragments were subjected to quantitative PCR using primers listed in Table 1. Four biological replications were included in each treatment.

After treatment by different concentrations of LPA, LPC or ZEA for 72 h, porcine GCs were collected for western blotting analysis according to standard methods [36 (link)]. The protein of each group was separated by SDS-PAGE and transferred to PVDF membranes. Then using 5% BSA dissolved in PBST with 0.05% Tween-20 (PH 7.4) to block for 1.5 h, the membranes were incubated with anti-GAPDH (ImmunoWay, YM3040, USA), anti-YAP (Bioworld, Q295), anti-ACTIN (Abcam, ab8226, USA) overnight, at 4 ºC. Followed be washing three times with TBST, the membranes were incubated with secondary antibodies (Beyotime, A0208 or A0216) at a dilution of 1:2000 in TBST. The band intensity was quantified using ACTIN or GAPDH as internal control and measured with IPWIN software.