Anti p akt antibody

Protein samples were extracted from tissues and cells with the same procedures described previously52 (link)53 (link). Briefly, heart tissues and HAECs were lysed in RIPA buffer and then centrifuged at 4 °C 13500 rpm for 15 minutes. The supernatant was collected, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein extracts were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked and incubated with the following primary antibodies: anti-VE-cadherin antibody, anti-CD31 antibody, anti-α-SMA antibody, anti-FSP1 antibody, anti-Snail antibody (Abcam, Cambridge, MA, USA), anti-p-AKT antibody, anti-AKT antibody, anti-p-GSK-3β antibody, anti-GSK-3β antibody (Wanleibio. Shenyang, China), and anti-GAPDH antibody (Kangcheng, Shanghai, China); GAPDH was used as a loading control. After incubation at 4 °C overnight, the membrane was washed three times with PBST, incubated with fluorescence-conjugated goat anti-rabbit IgG and goat anti-mouse IgG for one hour at room temperature (1:10000, Invitrogen) and scanned by Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA).

Matched pairs of primary HCC tissues and adjacent liver samples were used for the construction of a TMA (in collaboration with the Shanghai Biochip Company, Shanghai, China). The TMA construction was performed as previously described [21 (link)]. Immunostaining was performed on TMA slides following the routine protocol. Following antigen retrieval, the samples were incubated with 3% H₂O₂, blocked with goat serum and probed with anti-14-3-3β antibody (1:50;ABGENT) overnight at 4°C, followed by serial incubations with HRP-conjugated secondary antibody and diaminobezidine (Sigma-Aldrich) for chromogenic reactions. The samples were also probed for p-Akt, using anti-p-Akt antibody (1:50, Wanleibio), followed by reactions with alkaline phosphatase-conjugated secondary antibody and the chromogen substrate BCIP/NBT (Beyotime). Cell nuclei were counter-stained with hematoxylin.
The TMA slides were scanned with an Aperio ScanScope GL and assessed by the Aperio ImageScope software (Aperio Technologies, Vista, CA). Scoring of the TMA samples was based on the percentage of positively stained cells and the staining intensity. The scores equal to or above the median of all the values were defined as high, while the scores below the median were defined as low.