Sirt2

Frozen lung tissue was homogenized in mammalian protein extraction reagent (M‐PER) to prepare a protein sample. Lysates of BEAS‐2B cell were prepared similarly. The lysates were mixed with 10% SDS‐PAGE then electrophoresis was performed on the same volume. After the electrophoresis, the protein was transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL, USA) then blocked with 5% milk for 1 hr at room temperature. The proteins were incubated with Snail (1:1000; Cell Signaling Tech.), E‐cadherin (1:1000; Cell Signaling Tech.), Vimentin (1:1000; Cell Signaling Tech.), α‐SMA (1:1000; Cell Signaling Tech.), β‐actin (1:1000; Cell Signaling Tech.), Sirt1 (1:1000; Cell Signaling Tech.), Sirt2 (1:1000; Cell Signaling Tech.), Sirt3 (1:1000; Cell Signaling Tech.), Sirt6 (1:1000; Cell Signaling Tech.) antibodies at 4°C overnight in a shaker incubator. After washing with TBS‐T, the membranes were incubated with anti‐rabbit IgG horseradish peroxidase conjugated antibody (1:5000; Cell Signaling Tech.) for 1 hr at room temperature. The protein bands were visualized using enhanced chemiluminescence with a Super Signal west pico kit (Bridgen Biological Technology, Shanghai, China). Films were scanned and analysed by densitometry using Syngene GeneGenius software (Syngene, Frederick, MD, USA).

Cell lysates and co-immunoprecipitated samples were separated on 4–12% SDS–PAGE gels (Thermo Fisher Scientific) and then transferred to membrane using Bio-Rad Trans-Blot Turbo system. Membranes were blocked; incubated with primary antibodies—acetylated α-tubulin (6-11B-1; Santa Cruz Biotechnology), ATAT1 (ab58742; Abcam), SIRT2 (12650; Cell Signalling Technology), HDAC6 (H-300; Santa Cruz Biotechnology), PMCA2 (PA1-915; Thermo Fisher Scientific), and NPC1 (NB400-148; Novus Bioscience); washed, incubated with appropriate HRP-conjugated secondary antibody and developed with chemiluminescent substrate (Thermo Fisher Scientific). Images were captured on a Bio-Rad ChemiDoc XRS+ system and quantified using ImageLab software (Bio-Rad). Membranes were then stripped and re-probed with primary antibodies.

Cell lysates were obtained on ice using lysis buffer (Beyotime), followed by centrifugation at 16 000 g for 10 minutes. Thereafter, the lysate was heated at 95°C for 5 minutes in loading buffer. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (10%) was used to separate the proteins. After being transferred onto a nitrocellulose membrane (Merck Millipore), the separated proteins were blocked for 2 hours using 5% non‐fat milk or bovine serum albumin. Overnight incubation at 4°C was conducted after adding the primary antibody against SIRT2 (Cell Signaling Technology; dilution 1:1000) or β‐actin (Sigma‐Aldrich; dilution 1:2000). An HRP‐conjugated secondary antibody (Cell Signaling Technology; dilution 1:2000) was added to the membrane and incubated for 1 hours at room temperature. Then, the membrane was washed three times using Tris‐buffered saline containing Tween 20 (TBST). After incubation, additional rinses were performed using TBST. A Molecular Imager ChemiDoc XRS + Imaging System was used to scan the protein bands, and the Image Lab software (Bio‐Rad Laboratories) was used for quantification after enhanced chemiluminescence reagents (Thermo Fisher Scientific) were employed to detect chemiluminescence signals.