Anti p akt ser473 antibody

Protein lysate was extracted and the protein concentration was determined by the BCA method (Biyuntian, Nantong, China). Total 30 μg protein in each condition was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose membranes (Immobilon-P, Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk in TBST (10 mM Tris, pH 7.4, 150 mM NaCl and 1‰ Tween-20) at room temperature for 1 h and incubated with the anti-P-Akt (Ser473) antibody (1:1000 dilution, Cell Signaling Technology, Beverly, Mass, USA), anti-T-Akt antibody (1:1000 dilution, Cell Signaling Technology, Beverly, Mass, USA), pan anti-acetyllysine antibody (1:1000 dilution, PTM Biolabs, Hangzhou, China), anti-integrin β5 antibody (1:500 dilution, Abcam, MA, USA), anti-acetyl-Histone H2B (Lys12) antibody (1:2000 dilution, PTM Biolabs, Hangzhou, China), and anti-GAPDH antibody (1:5000 dilution, Kangchen bio-tech, Shanghai, China) at 4 °C overnight. After washing with TBST, the membranes were reacted with the peroxidase-conjugated affiniPure goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000 dilution, Zhongshanjinqiao, Beijing, China) for 1 h at room temperature. After extensive washing with TBST, signals were detected using enhanced chemiluminescent reagents (Thermo Scientific, IL, USA).

Immunostaining was performed as described [20 (link)]. Briefly, after fixation with 4% paraformaldehyde for 15 min, 3D HME1 acini were incubated with PBS + 0.2% Triton X100 for 10 min, followed by blocking with PBS + 1% BSA, 1% FBS and 0.05% Tween 20 for 1 h and incubation with the primary antibody overnight at 4 °C. Cells were then rinsed extensively with PBS, and incubated with appropriate secondary antibodies for 2 h, rinsed extensively with PBS, and stained with DAPI (Sigma, St Luis, MO, USA). Slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). ANXA8 was detected with an anti-ANXA8 antibody (LifeSpan BioSciences, Seattle, WA, USA) followed by anti-rabbit Alexa Fluor 488. P-AKT was detected with an anti P-AKT (Ser 473) antibody (Cell Signaling, Danvers, MA, USA) followed by anti-rabbit Alexa Fluor 546. 3D acini were analyzed with a confocal microscope (SP2 Spectral Confocal Microscope, Leica, Wetzlar, Germany).

The proteins of the LL sample were extracted using the method put forward by Li et al. (2017) (link). After the separation of proteins by SDS-PAGE (10 % separation gel and 5 % concentration gel), the target proteins (50 μg) were transferred onto polyvinylidene difluoride (PVDF) films (Millipore, Bedford, MA, America). Then, PVDF films were subjected to blockade in 5 % fat-free milk in Tween 20 (TBS-T) solution (0.1 % Tween-20, 150 mM NaCl, 10 mM Tris, 5 mM KCl, pH 7.4) under ambient temperature and then incubated for 12 h under 4 °C using a variety of different primary antibodies: anti-HIF-1α antibody (1:500), anti-PI3K antibody (1:500), anti-AKT antibody (1:500), anti-p-AKT (Ser473) antibody (1:500), and anti-Actin antibody (1:3,000) (Cell Signaling Technology, Beverly, MA, America). Posterior to cleaning three times (10 min per time) with TBS-T solution, the films were cultivated for 1 h under ambient temperature using anti-rabbit horseradish peroxidase-conjugated second antibody (1:5,000) (Abcam, Cambridge, UK). Then, the films were cleaned thrice in TBS-T solution again and the proteins were visualized using the ECL assay kit on a ChemiDoc MP imaging system (Bio-Rad, Hercules, America). The expressing levels of proteins were subjected to quantification via the Quantity-One program 4.6.2 (Bio-Rad, America) and afterward normalized to actin.