Ab32389

Protein samples were extracted from myocardial tissue and H9C2 cells. Protein concentrations were determined using the BCA method to ensure consistent loading of each sample. Proteins were separated using SDS-PAGE, and then transferred to 0.45-μm PVDF membranes. After blocking with skim milk for 1 hour, membranes were incubated with antibodies against Bcl-2 (ab32124), Bax (ab53154), p53 (ab32389), LC3B(ab63817), SQSTM1/62 (ab91526), ATG5 (ab53154), Beclin1 (ab62557), or β-actin (ab8226) (antibodies purchased from Abcam Biotechnology, USA) at 4 °C overnight. After labeling with secondary antibodies, the target protein was observed using enhanced chemical luminescence.

PBS at 4 °C was used to wash the cells twice. Thereafter, protease inhibitors in cold RIPA buffer were used to lyse the cells. Protein concentration was determined using BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China). Total protein of the cells were denatured using 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the membrane for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4 °C (dilution ratio 1:2000). TBST was used to wash the membranes three times and they were subsequently incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG) for 1 h at room temperature. TBST was once again used to wash the membranes three times. The targeted proteins were identified using the ECL (EMD Millipore, MA, USA) method. SKA3, CCNE2, CCNA2, CDK4, CDK2, P53, P53-pSer15, P53-pSer46 antibodies used for western blot in this research were purchased from Bioss (bs-7848R), Abcam (ab32147), Abcam (ab108357), Abcam (ab181591), Abcam (ab32103), Abcam (ab32389), CST #9284 and CST#2521.

The transplanted tumors were surgically removed until the tumor volume reached 100 mm³, and then fixed in 10% formalin. Then, the tumors derived from the donor’s pelvic lymph node and the transplanted tumors were subjected to routine hematoxylin and eosin (HE) staining and staining for other markers, including P16 (1:50, ab51243, Abcam, Cambridge, UK), P53 (1:2000, ab32389, Abcam), ER (1:800, 21244-1-AP, Proteintech), PR (1:200, 25871-1-AP, Proteintech, Chicago, IL, USA), PAX8 (1:200, 10336-1-AP, Proteintech), and WT1 (1:500, ab89901, Abcam). Microscopic slides were reviewed by a senior gynecology-dedicated pathologist (Prof. Yang).
Similarly, immunohistochemistry (IHC) was conducted to assess the expression of Ki67 (1:200, AF0198, Affinity, San Francisco, CA, USA), CyclinD1 (1:250, ab134175, Abcam), Hes1 (1:200, ab108937, Abcam), N-cadherin (1:200, #13116, Cell Signaling Technology, Danvers, MA, USA), vimentin (1:200, #5741, Cell Signaling Technology) and CSF3 (1:200, 17185-1-AP, Proteintech) in the tumors extracted from mice. The kidney and liver were also removed for HE staining to evaluate the toxicity of viscera.
Finally, the expression of CSF3 in the corresponding lymph nodes samples from 10 ovarian cancer patients was evaluated by IHC. The scoring criteria for staining intensity were described in our previous research [33 (link)].