Anti hdac6

RPMI culture medium was purchased from Corning (Corning, NY). Fetal bovine serum (FBS) was purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin, pen/strep antibiotics, and puromycin were purchased from Gibco (Grand Island, NY). Trametinib (MEK inhibitor) was purchased from Selleckchem (Houston, TX). Antibodies for Western Blot: Anti-HDAC1 (#2062, from Cell Signaling Technology (Danvers, MA), anti-HDAC2 (#2540 CST), The anti-HDAC3 and anti-HDAC8 antibodies were described in refs. 17 (link), HDAC11(#58442, CST). Anti-HDAC6 (#H-300, sc-11420) was purchased from Santa Cruz Biotechnologies. Cleaved PARP (Asp214) (#D64E10) (#5625 CST), phospho LKB1 (#3482, CST), total LKB1 (#3050, CST). Anti-YAP1 antibody (#H00010413-M01, Abnova). Anti-Vinculin (#G8796) and anti-GAPDH (#V9131) were purchased from Millipore Sigma (Bedford, MA).

Cells grown on coverslips were transfected with Twist1-GFP deletion construct and immunostained at desired time points using specific antibodies as mentioned below. Transfected cells were fixed in 3.7% formaldehyde (15 min), washed with PBS, permeabilized with 0.2% Triton X-100 in PBS (5 min) and then blocked with 3% BSA in PBS for at least 30 min at RT. The cells were washed once and specific primary antibodies [anti-Vimentin (Santa Cruz, Cat # SC-6260), anti-HDAC6 (Santa Cruz, Cat # SC-28386), and anti-γ tubulin (Sigma, Cat # T6557)] were added in a freshly prepared 0.5% BSA in PBS (1:500) and incubated for 2 h at RT. Following seven PBS washes, the secondary antibodies conjugated to Alexa Fluor 555 (Invitrogen, Cat # A-21422) were added (1:1000 in PBS) to the cells and incubation continued for 1 h at RT in dark. Finally, the cells were washed ten times with PBS and stained with DAPI. Finally, the coverslips were mounted on glass slide using mounting media Mowiol-DABCO and images were captured at different magnifications using Nikon TiS. Fluorescence pictures were merged using Image J software.