Sodium dodecyl sds polyacrylamide gel

Following cell treatments, the whole cell lysates were collected after mixing cell pellets with 100 μL of radio immune precipitation assay buffer (RIPA). Protein sample concentration was determined by using the bicinchoninic acid protein assay kit (Merck, Poole, UK). Each sample (20 μg) was electrophoresed through a 4–12% sodium dodecyl (SDS)-polyacrylamide gel (Life Technologies, Renfrewshire, UK), transferred onto a nitrocellulose membrane (Hybond-C, Amersham; UK), and probed with apoptotic antibodies (Danvers, MA, USA). GAPDH was used as a loading control (Merck, Poole, UK). Levels of protein expression were assessed using Pierce ECL Western blotting detection kit (Thermo Scientific; Leicestershire, UK). Membranes were scanned using benchtop G:box (Syngene, Cambridge, UK), and density was calculated using the ImageJ program (http://rsbweb.nih.gov/ij/ (accessed on 17 November 2022) incorporating correction of loading controls.

Following cell treatments, the whole cell lysates were collected after mixing cell pellets with 100 μL of radio immune precipitation assay buffer (RIPA). Protein sample concentration was determined by using the bicinchoninic acid protein assay kit (Pierce; USA). Each sample (20 μg) was electrophoresed through a 4–12% sodium dodecyl (SDS)-polyacrylamide gel (Life Technologies, UK), transferred onto a nitrocellulose membrane (Hybond-C, Amersham; UK), and probed with antibodies which include CD45 (PA5-95,187), JAK2 (MA5-15,632), ACTR2 (A305-216A-M), THAP3 (PA5-50,841), PBX-1 (PA5-29,674), and SEG (PA5-51,462), from ThermoFisher Scientific, UK. Other apoptotic antibodies such as caspase 3 (9662S), caspase 9 (9502S), cleaved caspase 9 (7237S), PARP (9542S), and cleaved PARP (5625S) were purchased from Cell Signaling, UK. Rabbit anti-rat HRP (ab6734) and rabbit anti-mouse HRP (58802S) antibodies were purchased from Abcam-UK. CRISPR-Cas9 (NBP2-36,440) was purchased from Novus. GAPDH was used as a loading control (Sigma; UK). Levels of protein expression were assessed using Pierce ECL western blotting detection kit (Thermo Scientific; UK). Membranes were scanned using benchtop G:box (Syngene; UK) and density was calculated using the image J program (http://rsbweb.nih.gov/ij/) incorporating correction of loading controls.