Ab240639

IHC was performed as described previously [80 (link)]. Paraffin-embedded clinical tissues were incubated with the following antibodies: anti-SOX9 (abcam, ab185966; 1:1000 dilution), anti-RUNX2 (abcam, ab192256; 1:1000 dilution), anti-TTF1 (abcam, ab76013; 1:250 dilution), anti-Ki67 (Proteintech, 27309-1-AP; 1:5000 dilution), anti-SOX4 (abcam, ab243041, 1:1000 dilution), anti-RUNX1 (abcam, ab240639, 1:2000 dilution), anti-SIX1 (abcam, ab252224, a:100 dilution), and anti-Clusterin (abcam, ab92548; 1:200 dilution). The immunostaining was reviewed and scored blindly. The scoring system for grading expression level was reported previously [81 (link)]. The score of each sample was multiplied by the grading of intensity and staining area.

CD4⁺ T cells and CD19⁺ B cells from mice were lysed through the RIPA buffer (WB038, GEFAN, China) [18 (link)]. The extracted protein was centrifuged and then quantified with the help of the BCA kit (23225, Thermo Scientific, USA). After being electrophoresed, they were transferred to the PVDF membrane before being blocked with 5% skim milk. Next, we utilized primary antibodies to treat the membrane at 4℃ all night. After that, second antibody was used to treat the membrane at 37℃ for 90 min. After rinsing, the reactive bands were reacted with the color reagent (34075, PIERCE, USA) in a gel imaging system (610020-9Q, Clinx, China). The primary antibodies of RUNX1 (1:1,000, ab240639, 48 kDa), FOXP3 (1:1,000, ab20034, 50 kDa), Bax (1:8,000, ab32503, 21 kDa), Bcl-2 (1:2,000, ab196495, 26 kDa), cleaved caspase-3 (1:5,000, ab214430, 17 kDa), caspase-3 (1:2,000, ab184787, 32 kDa), and GAPDH (1:10,000, ab181602, 36 kDa), and the second antibodies of goat anti-rabbit IgG H&L (HRP) (1:2,000, ab205718) and goat anti-mouse IgG H&L (HRP) (1:2,000, ab205719) were gained from Abcam (UK). GAPDH was exploited to the housekeeping gene. The protein level was normalized to GAPDH, and presented relative to control cells (set as 1).

Cells were lysed in Radioimmunoprecipitation assay buffer (Millipore) containing protease and phosphatase inhibitors (Thermo Fisher Scientific) to extract the total protein. Protein concentrations were determined using a bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific). Western blotting analysis was performed as described in our previous study (16 (link)). The antibodies used in this study recognized VIRMA (ab271136, 1:1000, Abcam), IG2BP2 (ab188200, 1:2000, Abcam), E2F7 (ab245655, 1:1000, Abcam), ITGA2 (ab181548, 1:1000, Abcam), ITGA5 (ab150361, 1:1000, Abcam), NTRK1 (ab76291, 1:1000, CST), protein kinase B (Akt; #4685,1:1000, CST), phospho-Akt (#4060, Ser473, 1:1000, CST), CBFB (ab133600, 1:1000, Abcam), RUNX1 (ab240639, 1:1000, Abcam), and α-tubulin (ab7291, 1:5000, Abcam).