Anti chi3l1

RIPA buffer supplemented with protease inhibitor cocktail (Pierce, IL, USA) was used to lyse cells, after which nuclear and cytoplasmic proteins were isolated with a commercially available nuclear and cytoplasmic extraction kit (Thermo Fisher Inc., IL, USA) based on provided instructions. A BCA assay (Beyotime Biotechnology, China) was then used to measure protein levels, and equal amounts of protein (40μg) per sample were separated via 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Germany) that were then blocked using 5% non-fat milk in TBST for 1 h. Next, blots were probed overnight with anti-Nrf2 (1:1000, Abcam), anti-CHI3L1 (1:200, Santa Cruz), anti-LaminB (1:1000, Sigma), anti-α-tubulin (1:1000, Santa Cruz), or anti-GAPDH (1:1000, Santa Cruz) at 4°C. Blots were then washed using TBST and probed with an appropriate secondary antibody for 2 h at room temperature, after with the ECL Plus reagent (Amersham Biosciences, Little Chalfont, UK) and autoradiographic film were used to detect protein bands. A gel image analysis system (BioRad, CA, USA) was used for densitometric analyses of developed blots.

Tissue sections were blocked for 1 h using 5% goat serum at room temperature, and were then incubated overnight with anti-Nrf2 (1/500, Abcam) or anti-CHI3L1 (1/50, Santa Cruz, USA). Sections were then washed thrice with PBS and stained for 1 h with secondary goat anti-mouse AF488 (1/200, Invitrogen, USA) and rhodamine-conjugated goat anti-rabbit IgG (1/100, Abcam). Nuclei were then stained with DAPI, and cells were imaged via laser-scanning confocal microscopy (Olympus, Tokyo, Japan).