Anti plek2

Bone marrow and FLC-derived CD45⁻ erythroblasts were lysed with protease inhibitors (Protease Inhibitor Cocktail Tablets, Roche), quantified, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10–12% polyacrylamide gels, and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h in TBST (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.05% Tween 20) containing 5% skim milk or 5% BSA, followed by overnight incubation with commercially available primary antibodies (anti-plek2 1:1000 (Proteintech, 11685-1-AP); anti-cofilin 1:1000 (Santa Cruz, sc-376476); anti-Rac1 1:1000 (Cell Signaling Technology, 2465); anti-VDAC1 1:1000 (Millipore, MABN504); anti-cytochrome c 1:1000 (Abcam, ab90529); and anti-GAPDH 1:4000 (Invitrogen)). Blots were washed and incubated for 1 h at room temperature (RT) with the secondary antibody (horseradish peroxidase (HRP) conjugated (Thermo Scientific)). Immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) method (Amersham Bioscience) according to standard procedures. For mitochondria fractionation and activated Rac1 pull-down (Thermo Scientific, 89,874 and 16,118, respectively), samples were processed according to the manufacturer’s instructions.

Cells were transfected with the indicated constructs for 48 h and then cells were dissolved in IP lysis buffer (Thermo Fisher, Inc) with protease inhibitor cocktail (Sigma) and PMSF (Sigma) for 1 h at 4 °C. After centrifugation at full speed for 20 min, cleared supernatant were gently rotated with antibodies and protein A beads (Invitrogen) for 4 h at 4 °C. Then beads were washed four times with IP lysis buffer. The beeds were eluted in 1X SDS buffer. Primary antibodies were as followings: anti-PLEK2 (Proteintech), anti-EGFR (Cell Signal Technology), anti-IgG (Abcam), and anti-Flag (Sigma).

All regents including Cisplatin (Sigma, p4394) and Recombinant Human TGF-β1 (R&D, 240-B/CF) were obtained from commercial sources. The following antibodies were used: anti-PLEK2 (1:200 for IHC, 1:500 for Western blot, Proteintech, 11685-1-AP), anti-α-Tubulin(1:5000, Sigma, T9026), anti-Akt (1:1000, CST, #9272), anti-p-Akt Antibody(Ser 473)(1:1000, CST, #4060), anti-Erk (1:1000, CST, #4695), anti-p-Erk (1:500, CST, #4370), anti-Claudin-1 (1:500, CST, #13255), anti-Cyclin B1 (1:1000, CST, #4118), anti-Cyclin E2 (1:1000, CST, #4132), anti-Bax (1:1000, CST, #14796), anti-Bcl-2 (1:!000, CST, #3498), anti-PARP (1:1000, CST, #9532), anti-Cleaved-PARP (1:1000, CST, #9548), anti-Caspase 3 (1:1000, CST, #9662).