Jak2 17670 1ap

Cells were harvested, and 1 × RIPA buffer containing a protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) was added on ice for 30 min and centrifuged at 12,000 g for 15 min at 4 ℃ to pellet cell debris. Protein concentration was determined using a BCA reagent (Thermo Fisher Scientific, USA). After heating to 100 ℃ for 10 min, the protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membrane was blocked with 5% powder milk and probed with an antibody at 1:1000. After washing with 1 × TBST buffer, the membrane was incubated with HRP-conjugated secondary antibodies at 1:5000. The target proteins were visualized using an ECL kit (Thermo Fisher Scientific, USA) and imaged using a Bio-Rad imaging system. Antibodies against GAPDH #8884, STAT3 #4904, STAT5 #94205S, Phospho-STAT5 #9359, Phospho-BCL2 #2827S, Caspase3 #9662S, PARP #9532S were purchased from CST (Danvers, MA). Antibodies against SPATS2L 16938-1-AP, JAK2 17670-1AP, BCL2 26593-1-AP were purchased from ProteinTech (Rosemont, USA). The antibody against CD45 (PE conjugated) was purchased from MULTI SCIENCES (Hangzhou, China).

The primary antibodies are PD-L1 (CST #13684, Cell Signaling Technology), PD-L1 (17952- 1-AP, Santa Cruz), STAT3 (CST #9139, Cell Signaling Technology), phosphorylated STAT3 (CST #9145, Cell Signaling Technology), JAK2 (17670-1-AP, Proteintech), phosphorylated JAK2 (CST #4406, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), phosphorylated AKT (66444-1-Ig, Proteintech), ERK (CST #4696, Cell Signaling Technology), phosphorylated ERK (CST #3510, Cell Signaling Technology), PARP-1 (sc-8007, Santa Cruz), and GAPDH (60004-1-Ig, Proteintech). Goat anti-rabbit antibody and mouse anti-rabbit antibody conjugated to HRP were purchased from Biosharp. all antibodies and reagents were stored and used according to the manufacturer’s instructions. Briefly, tissues were homogenized, mixed with 5X loading buffer and boiled until denaturation. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes are sealed with 5% skim milk and incubated overnight at 4°C with the primary antibody. The membranes were then washed, incubated with secondary antibodies for 1 hour at room temperature, and visualized with SuperSignal West Pico plus chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA).