Akap 9

Cell lysates were prepared using SDS lysis solution. Protein concentrations were measured using a BCA protein assay kit. Equal amounts of protein were separated by electrophoresis on 10% SDS-polyacrylamide gel and then electrotransferred from the gel to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk solution for 1 h and then incubated with primary monoclonal antibodies against AKAP-9 (Abcam), SRPK1 (Abcam) and SRSF1 (Abcam) overnight at 4°C. Phospho-SRSF1 was incubated with mAb1H4 antibody [29]. α-Tubulin was used as an internal control. After washing with TBS-T, the membrane was incubated with secondary antibodies against goat or mouse IgG. The membrane was then washed, and blots were detected using an enhanced chemiluminescence (ECL) detection system (Thermo) according to the manufacturer's instructions.

Cells were harvested by scraper and lysed with Triton buffer 150 mM NaCl, 1% Triton-X100, 0.1% SDS, 50 mM Tris pH8.0, and protease inhibitor cocktail (Thermo Fisher) at 4°C for 20 min. The supernatant was collected after centrifugation for 10 min. Supernatant was transferred to a new tube and the total protein concentration was measured using NanoDrop One spectrophotometer. Equal volume 2X Laemmli sample buffer (1610737, Bio-Rad, Hercules, CA) were added to the supernatant and then heated in 95°C for 10 min. Equal amount proteins were resolved by SDS-PAGE gel for western blot analysis. Antibodies against AKAP9 (1 : 1000), GAPDH (1 : 3000), E-cadherin (1 : 2000), and Rabbit secondary antibody (1 : 5000) were purchased from Abcam (Cambridge, MA). The western blot images were developed using chemiluminescence detection kit (WBKLS0500, Millipore, Billerica, MA) and the ChemiDoc Imaging System from Bio-Rad.