Ab182579

Cells (A549-pSin-Vector, A549-SOX9, NCI-H460-pSin-Vector, NCI-H460-SOX9, A549-pSuper-Vector, A549-SOX9 sh1#, NCI-H460-pSuper-Vector and NCI-H460-SOX9 sh1#) for immunofluorescence staining were grown and treated in chamber slides, fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized for 10 min with 0.2% Triton X-100 in PBS, and blocked with 2% bovine serum albumin (BSA) for 1 h. Primary antibodies against SOX9 (#ab182579, Abcam), E-cadherin (#610181, BD Biosciences), γ-catenin (#610254, BD Biosciences), N-cadherin (#610920, BD Biosciences), vimentin (#550513, BD Biosciences), and β-catenin (#610154, BD Biosciences) were diluted to 1:400 in PBS containing 1% BSA and incubated for 1 h at room temperature. Secondary antibody was purchased from Life Technologies^® (Grand Island, NY), diluted to 1:250 in 1% BSA in PBS and incubated for 1 h. Images were captured with the Nikon^® TS2 microscope.

Total protein extraction was carried out by lysing cells with 1× SDS loading buffer and denatured at 95°C for 5 min. The sample volume per lane was 10 μL. The protein samples were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, USA). After being blocked and washed, the membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with the corresponding secondary antibodies. Finally, the membrane was exposed with ECL developer (CW0049M; CWbio, Beijing, China) and processed with ImageJ software (
https://imagej.nih.gov/ij/). The primary antibodies used were as follows: anti-GAPDH (1:1000; CWbio), anti-HDAC6 (1:1000; 12834-1AP; Proteintech), anti-acetylated tubulin (1:1000; T7451; Sigma, St Louis, USA), anti-α-tubulin (1:1000; T6199; Sigma), anti-collagen II (1:1000; ab34712; Abcam), anti-MMP13 (1:1000; Wanleibio, Shenyang, China), and anti-SOX9 (1:1000; ab182579; Abcam). The secondary antibodies were goat anti-mouse IgG (1:4000; CWbio) and goat anti-rabbit IgG (1:4000; CWbio).