Anti ef1α antibody

1 × 10⁷ BSF parasites were lysed in sample buffer (NuPAGE sample buffer) and analyzed on a 4–12% NuPAGE Novex BisTris gel using MES buffer. Subsequently, the samples were electroblotted on nitrocellulose membrane (GE Healthcare) and probed with the respective antibodies. The PNT1HA cell line was probed with anti-HA antibody (1:4000, Roche Applied Science). The PNT1Myc cell line was probed with anti-Myc antibody (1:2000, Millipore). Anti-EF1α antibody (1:5000, Millipore) was used for the loading controls.

Cell lysates were prepared using RIPA buffer (Millipore) and separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. Nuclear proteins were isolated using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology). The following primary antibodies were used: anti-FAM83B (1:1000, PA5-56754, Thermo), c-myc (1:1000, #18583, CST), cyclin D1 (1:1000, #18583, CST), BCL2 (1:1000, #4223, CST), Cleaved Caspase-3 (1:1000, #9661, CST), β-catenin (1:1000, #8480, CST), Flag (1:1000, #14793, CST) and His (1:1000, #12698, CST) antibodies. Anti-GAPDH antibody (1:1000, #5174, CST) was used as the loading control for total proteins, anti-EF-1α antibody (1:1000, 05-235, Millipore) was used as the loading control for nuclear proteins.
Total RNA was isolated using TRIzol regent (Thermo) and was reversely transcribed with HiScript Reverse Transcriptase (Vazyme) according to the manufacturer’s instructions. Q-PCR was carried out with using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa) according to the manufacturer’s instructions on a CFX96 Touch Real-time PCR Detection system (Bio-Rad). GAPDH was used for the normalization of the Q-PCR.