Anti pim1

Samples were separated on a 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose membranes and then blocked in 10% dry milk‐TBST (20 mmol/L Tris‐HCl [PH 7.6], 127 mmol/L NaCl, 0.1% Tween‐20) for two hours at 37°C. Following three washes in Tris‐HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with primary antibody overnight at 4°C. Following three washes, membranes were then incubated with secondary antibody overnight at 4°C. Primary antibodies include the following: anti‐glypican‐3 (Abcam), anti‐PCNA (Abcam), anti‐METTL3 (Abcam), anti‐histone H3 (Abcam), anti‐H3K9me3 (Abcam), anti‐JMJD2A (Santa Cruz, Biotech), anti‐Pim1 (Santa Cruz, Biotech), pHistone H3 (Abcam), anti‐SUZ12 (Santa Cruz, Biotech), anti‐SUV39H1 (Santa Cruz, Biotech), anti‐Nanog (Abcam), anti‐MEKK4 (Abcam), anti‐pTyr (Abcam). Signals were visualized by enhanced chemiluminescence plus kit (GE Healthcare) according to our pervious protocol.34

Cells were lysed in 2x LSB and heated at 95 °C for 5 min. Proteins were separated by SDS-PAGE, immobilized onto PVDF-membrane (EDM Millipore, Merck) and incubated overnight with anti-PIM-1 (1:500, 12H8; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PIM-2 (1:1000, D1D2; Cell Signaling Technology, Danvers, MA, USA), anti-PIM-3 (1:1000, D17C9; Cell Signaling Technology), anti-NFATC1 (1:500, Santa Cruz Biotechnology), anti-V5 (1:500, Invitrogen, Carlsbad, CA, USA), anti-Flag (1:500, F1804; Sigma-Aldrich), anti-ACTB (anti-β-actin; 1:1000, 13E5, #4970S, Cell signaling Technology), anti-GAPDH (1:50000, Sigma-Aldrich), anti-β Tubulin (1:40000, Sigma-Aldrich) or anti-Fibrillarin (1:1000, Cell Signaling Technology) antibodies. After incubations with secondary antibodies, chemiluminescence reactions were generated using either Amersham™ ECL Plus or ECL Prime reagents (GE Healthcare).