Odyssey clx two color infrared laser imaging system

Hela and Siha cells were collected and lysed with cell lysis buffer for western blot and IP (Beyotime, China) supplemented with protease inhibitor cocktail (APExBIO, USA) to harvest proteins. Cells were lysed on ice for 30 min, and the lysate was obtained by centrifugation at 12,000 rpm for 15 min. Proteins were fractionated by SDS-PAGE, and then transferred onto 0.45 μM PVDF membranes. The PVDF membranes were blocked with 5% nonfat milk in TBS/Tween-20, and blotted with specific antibodies at 4°C overnight. The antibodies used for western blot are as follows: YTHDF1 (ProteinTech, 1:1000), RANBP2 (ProteinTech, 1:1000), GAPDH (ZSGB-BIO, 1:2000), Flag-tag (MBL, 1:5000). After washed with TBS/Tween-20, the PVDF membranes were incubated with fluorescent secondary antibody (LI-COR, IRDye 680RD Goat anti-Rabbit and IRDye 800CW Goat anti-Mouse, 1:10000). ODYSSEY Clx Two-color infrared laser imaging system (LI-COR, USA) was used to visualize the bands.

Total proteins were firstly extracted from cells, which were washed by cold PBS and treated with RIPA lysis buffer (KeyGEN, KGP703) (supplemented with protease inhibitors 1 mmol/L phenylmethylsulfonylfluoride, 10 mg/L pepstatin, 10 mg/L aprotinin and 5 mg/L leupeptin). Protein concentrations were determined using the BCA protein assay regents (#23225, Thermo Pierce, Rockford, IL, USA). The procedure of western blot was conducted as previously described (9 (link)). Primary antibodies used in the study include anti-AKAP13 (Immunoway, YT0161; Abcam, ab99377), anti-HEG1 (Biorbyt, orb157480), anti-caspase 3 (Immunoway, YT6113), anti-caspase 3 p17 (Immunoway, YT6161). Anti-β-tubulin (Abcam, ab210797) was used as internal standard. Detection was achieved in Odyssey CLX Two-color infrared laser imaging system (LI-COR Biosciences, Nebraska, USA). Densitometric analysis of the bands was performed using ImageJ software.

Total proteins, nuclear proteins, or cytoplasmic proteins were extracted using the cell lysis buffer (Cell Signaling Technology, USA), nuclear protein and cytoplasmic protein extraction kit (Beyotime, China), respectively, according to the manufacturer’s instructions. Protein concentration was measured using a BCA protein assay kit (Beyotime, China). Total proteins, nuclear proteins, or cytoplasmic proteins were separated by 10–12% SDS-PAGE (BOSTER, China) and transferred to PVDF membranes (Bio-Rad, USA). After blocking with 5% nonfat milk (BD Biosciences, USA) for 1 h, the membranes were incubated with primary antibody and 5% nonfat milk overnight at 4 °C (the dilution ratio of primary antibodies is 1 : 1000). The membranes were then washed three times with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05%V/V Tween-20) and incubated with the corresponding secondary antibody for 1 h (the dilution ratio of secondary antibodies is 1 : 20,000). Images were performed using the Odyssey CLX two-color infrared laser imaging system (LI-COR Biosciences, USA). The densitometric analysis of blots was performed by Image Studio Ver 5.2 software (LI-COR Biosciences, USA). The values were normalized to those of control β-actin or GAPDH (for total protein, cytoplasmic protein), or PCNA (for nuclear protein).