Anti srsf5

Total cellular proteins were extracted using RIPA lysis buffer (Beyotime), separated via electrophoresis on a 10% SDS separation gel (YEASEN, China), and transferred to PVDF membranes (Millipore, Germany). The PVDF membranes were then incubated with primary antibodies, including anti-SRSF5 (1:1000; Abcam, UK), anti-HOXD8 (1:1000; Santa Cruz, USA), anti-TRIM72 (1:2000; Proteintech, USA), anti-PKM2 (1:1000; CST, USA), and anti-GAPDH (1:5000; ABclonal, China) antibodies at 4 °C. Subsequently, the membranes were incubated with Peroxidase AffiniPure Goat Anti-Rabbit/Mouse IgG (H + L) (1:10000; Jackson ImmunoResearch, USA) for 1 h at room temperature. The protein bands were visualized with a SuperPico ECL Chemiluminescence Kit (Vazyme) and imaged using a ChemiDoc XRS + Imaging System (Bio-Rad, USA).

The protein levels of SRSF5, PKM2 and HOXD8 were evaluated via IHC. After baking, dewaxing and hydration, the paraffin-embedded tissue sections were subjected to antigen retrieval and blocking. Subsequently, the sections were incubated with anti-SRSF5 (1:100; Abcam), and anti-HOXD8 (1:150; Santa Cruz) antibodies at 4 °C overnight. The next day, the sections were incubated with secondary antibodies, and staining was visualized using the GTVision III Detection System/Mo&Rb (GeneTech, China).