Akt 60203 2 ig

About 10 neurospheres were plated in a 60mm dish and subjected to
ISP or scrambled peptide for 21 hours. NSCs were homogenized with RIPA lysis
buffer and protein concentration was determined by Pierce BCA protein assay
kit according to the manufacturer’s instructions (#23227, Thermo
Fisher). Then, equal amounts of protein were loaded onto 15% or 12% SDS-PAGE
gels, and electrophoretically transferred to PVDF membranes (Millipore). The
membranes were blocked in 0.1% TPBS buffer with 5% BSA for 1 h at room
temperature and incubated with indicated primary antibodies overnight at
4°C and followed by secondary antibodies conjugated to horseradish
peroxidase. The following primary antibodies were used: AKT (#60203-2-Ig,
Proteintech), ERK1/2 (#16443-1-AP, Proteintech), p-Akt (Ser473) (# 4060S,
Cell signaling), GAPDH (#60004-1-Ig, Proteintech), p-ERK1/2 (Thr202/Tyr204)
(# 4370S, Cell signaling). Enhanced chemiluminescence was performed with a
West Pico Kit (Thermo Fisher). The density of bands was quantified using
ImageJ software (NIH). This experiment was repeated three times and each
pair of protein samples (scrambled peptide or ISP) were used for Western
blot quantification.

One rat testis per rat was removed and fixed in Bouin’s solution for 24 h. The testes were then dehydrated in graded ethanol, embedded in paraffin, and sectioned (4 µm in thicknesses). The sections were stained with hematoxylin-eosin (HE) dye and observed under a light microscope (Leica DM500, 100x).
Next, the sectioned paraffin-embedded slides of rats’ spermary per group were deparaffinized and rehydrated with xylene and gradient alcohol. The slides were then incubated with primary antibodies [PI3K p85 (60225-1-Ig, Proteintech), p-PI3K p85 (AF3242, Affinity), AKT (60203-2-Ig, Proteintech), p-AKT (AF0832, Affinity), JNK (66210-1-Ig, Proteintech), p-JNK (AF3318, Affinity), p53 (60283-2-Ig, Proteintech), and p-p53 (AF3075, Affinity)] overnight at 4°C following an antigen repair and closure. Next, the slides were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) at room temperature for 1 h. The slides were then treated with 3,3-diaminobenzidine (DAB) and hematoxylin, dehydrated with alcohol, and sealed with neutral gum. Finally, slides’ images were captured by the Olympus BX53 fluorescence microscope (40x).

MAT (purity: ≥ 98%) was purchased from Shanghai Yuanye Bio-Technology Co., LTD, (China) and dissolved in phosphate buffered saline (PBS). MDA-MB-231 and MDA-MB-468 cell lines were obtained from American Type Culture Collection (ATCC, United States). PI3K (67071-1-Ig), P-AKT (66444-1-Ig), AKT (60203-2-Ig), and GAPDH (60004-1-Ig) were purchased from Proteintech (Wuhan, China) PGK1 (sc-130335) and LC3- II (sc-271625) were purchased from Santa Cruz Biotechnology, Inc., (Texas, United States), and BCL-2 (ab182858) and cleaved caspase-3 (ab32042) were purchased from Abcam (Cambridge, UK).