Anti akts473

Cells were washed with ice-cold PBS and centrifuged at 400 × g for 5 min at 4 °C. The resulting pellet was lysed with RIPA buffer (KeyGEN) supplemented with 1x protease/phosphatase inhibitor (Solarbio). The homogenate was cleared by centrifugation at 4 °C for 10 min at 16,000 × g, and the supernatant containing the protein fraction recovered. Samples were subjected to electrophoresis in SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked with 1% non-fat milk and incubated with primary antibodies overnight, followed by 1-hour incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature and washed 3 times. The following primary antibodies were used: anti-p-p38 MAPK (Cat.#YP0338), anti-p38 (Cat.#YT3513) (Immunoway), anti-AKT (S473) (Cat.#A5030), anti-AKT (Cat.# 10176-2-AP) (Cell Signalling Technology), anti-CDK1 (Cat.# CY516-50), anti-GAPDH (Cat.# 12231 P) (Abways), and anti-cyclin B1(Santa Cruz Biotechnology). Antigen-specific binding of antibodies was detected with the ECL (enhanced chemiluminescence) Plus reagents (Beyotime). Protein bands were quantified ImageJ software (Wright Cell Imaging Facility) and then normalized to the GAPDH loading control. For full scans of Western blots see Supplemental Figures S1–S2.

Cells were washed with ice-cold PBS and centrifuged at 400 × g for 5 min at 4 °C. The resulting pellet was lysed with RIPA buffer (KeyGEN) supplemented with 1x protease/phosphatase inhibitor (Solarbio). The homogenate was cleared by centrifugation at 4 °C for 10 min at 16,000 × g, and the supernatant containing the protein fraction recovered. Samples were subjected to electrophoresis in SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked with 1% non-fat milk and incubated with primary antibodies overnight, followed by 1-hour incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature and washed 3 times. The following primary antibodies were used: anti-p-p38 MAPK (Cat.#YP0338), anti-p38 (Cat.#YT3513) (Immunoway), anti-AKT (S473) (Cat.#A5030), anti-AKT (Cat.# 10176-2-AP) (Cell Signalling Technology), anti-CDK1 (Cat.# CY516-50), anti-GAPDH (Cat.# 12231 P) (Abways), and anti-cyclin B1(Santa Cruz Biotechnology). Antigen-specific binding of antibodies was detected with the ECL (enhanced chemiluminescence) Plus reagents (Beyotime). Protein bands were quantified ImageJ software (Wright Cell Imaging Facility) and then normalized to the GAPDH loading control. For full scans of Western blots see Supplemental Figures S1–S2.

Adherent cells were lysed by RIPA lysis buffer (CWBIO) containing protease inhibitors (CWBIO) on ice for 30 min. BCA kit (Thermo Fisher) was used for protein concentration determination. Detailed protocols of Western blot had been described previously.
³⁴ (link) Primary antibodies in this study included anti‐DKK1 (1:1000, ab109416, Abcam), anti‐GAPDH (1:5000, ab8245, Abcam), anti‐pan‐Akt (1:1000, 4691S, Cell Signaling Technology), and anti‐Akt‐S473 (1:1000, 4060T, Cell Signaling Technology). The secondary antibodies included goat anti‐rabbit IgG H&L (HRP) (1:5000, ab6721, Abcam) and goat anti‐mouse IgG H&L (HRP) (1:5000, ab6789, Abcam). Images were captured by photo system (Mini Chemi 910, SinSage).

Cell number quantification was done with crystal violet [28 (link)]. Foci assays were performed seeding 500 cells per well (6-well plate) and staining and counting them by crystal violet. Lentiviral and retroviral transductions were performed as previously described [25 (link)]. Western blot was performed as previously described [29 (link)]. The following antibodies were used for Western blotting: rabbit panti-RpS6^S240/244, anti-RpS6, anti-HSP90, anti-PTEN (138G6), anti-AKT^S473, anti-AKT (all dilution 1:1000) and anti-cleaved PARP (D64E10)(dilution 1:1000) were all from Cell Signalling. RNA was extracted using NucleoSpin® RNA isolation kit from Macherey-Nagel (ref: 740955.240 C). 1 μg of total RNA was used for cDNA synthesis using qScript cDNA Supermix from Quanta (ref. 95048). Quantitative Real Time PCR (q-RTPCR) was performed as previously described [29 (link)]. Applied biosystems TaqMan probes: Gnmt/GNMT (Mm00494688_m1, Hs00219089_m1), FOXO1 (Hs00231106_m1) GAPDH/Gapdh (Hs02758991_g1/Mm99999915_g1), and B-ACTIN (Hs 99999903 m1).