Pakt ser473 antibody

Target modulation was assessed using qPCR for Axin2 (a marker of WNT974), and western blot for porcupine and phospho-Akt Ser 473 (a marker of buparlisib activity). qPCR was performed using Taqman assays (Life Technologies). Protein was isolated after 48 h of treatment in biological triplicates using radioimmunoprecipitation assay buffer and quantified with bicinchoninic acid assay (Thermo Fisher). Protein was run on bis-tris gels (Life Technologies) and transferred to polyvinylidene fluoride membrane. Primary pAKT-Ser 473 antibody (Cell Signaling Technology, Beverly, Massachusetts), β-actin (Cell Signaling Technology), and PORCN (Abcam, Cambridge, England) was incubated overnight with subsequent 1-hour incubation with horseradish peroxidase-conjugate secondary antibody. Staining was performed using a chemiluminescent substrate (ThermoFisher). Imaging was performed using an LAS-4000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). Quantification of blots was performed using ImageJ (NIH) and statistical analysis using GraphPad.

CYC31 was synthesized according to its natural structure in our lab [22 (link)]. Recombinant human PTP1B protein was also purified in our lab [18 (link)]. I-OMe AG538, insulin, palmitate, MTT, and fatty acid-free BSA were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2-NBDG was obtained from Invitrogen (Carlsbad, CA, USA). DMEM, fetal bovine serum, and horse serum were bought from Hyclone (South Logan, UT). Penicillin-streptomycin solutions were purchased from Millipore (Billerica, MA, USA). ECL Western Blotting Substrates were bought from Bio-Rad (Hercules, CA, USA). pIRS1 (Tyr632) antibody and GLUT4 antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pAkt (Ser473) antibody, pIRβ (Tyr1146) antibody, insulin Receptor β antibody, and IRS1 antibody were bought from Cell Signaling Technology (Danvers, MA, USA). β-actin antibody was obtained from Abcam (Cambridge, MA, USA). RNAiso Plus, PrimerScript^TM RT reagent Kit, and SYBR Premix ExTaq^TM Ⅱ were obtained from Takara (Dalian, Liaoning, China).

Poorly differentiated liver cancer cell line Mahlavu cells were cultured in 100 mm culture dish for 24 h. Growth medium was then replaced with IC₅₀ concentrations of chalcones 1, 9 and 11 or DMSO (control) supplemented medium. cells were incubated for 24 hours then were scraped and collected for western blot analysis. Anti-PARP antibody (Cell Signaling, 9532), pAkt (Ser473) antibody (Cell Signaling, 9271), Akt antibody (Cell Signaling, 9272), pNFkB-p65 (Ser468) antibody (Snat Cruz, sc101750), p21 antibody (Millipore, 05345), pRb (Ser807/811) antibody (Cell Signaling, 9308), Rb antibody (Santa Cruz, sc102), actin antibody (Sigma, A5441) and calnexin antibody (Sigma, C4731) were used as primary antibodies. Anti-rabbit (6154) and anti-mouse (0168) secondary antibodies were used. ImageJ tool (^{42 (link)}) used for protein band intensity comparison. Original Full length blots are given as Supplementary Information.

TMAs were assembled at the Brigham and Women's hospital at Boston, MA. The TMA had total 213 tumor samples, 196 normal lung samples and 14 control samples. A minimum of three tissue cores with a diameter of 1 mm was arrayed into a recipient block using an automated tissue microarrayer ATA-27 (Beecher Instruments, Sun Prairie, WI). All tissues were obtained under protocols approved by applicable IRBs and they were used for PTEN, and p-AKT staining. Tumor tissue immunohistochemistry (IHC) staining was performed using standard techniques as described previously [25] (link). All slides were reviewed by two independent pathologists who were blinded to the identity of the tissues. Two measurements, the percent and intensity of IHC staining were used to evaluate the level of protein expression in a tissue sample. The final IHC score was obtained by a semiquantitative method that accounts for staining intensity and percentage of cells stained. Scoring of the immunostaining in the nuclei was performed as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; and 3+, strong staining.
For IHC staining the p-AKT ^Ser473 antibody (Cell signaling, cat. # 4060) was diluted 1–20.

48 h after lentivirus infection, total protein was extracted from cells by RIPA cell lysis buffer with proteinase and phosphatase inhibitors. The supernatant of lysates was collected and electrophoresed in 10%-12% SDS-PAGE gel, then transferred onto PVDF membranes. Membranes were blocked in Tris-buffered saline plus 0.1% Tween-20 (TBST) with 5% non-fat dry milk or bovine serum albumin (BSA) at room temperature for 1h, followed by primary antibody incubation at 4°C overnight. On the next day after washing with TBST, membranes were incubated with secondary antibodies labeled with HRP at room temperature for 1 h. Then proteins were detected by ECL detection system (P1010, Applygen Technology, Beijing, China). Protein levels were measured by Image J software. Primary antibodies were listed as follows: caspase-3(No. 9662, 1:1000, Cell Signaling Technology), cleaved caspase-3 (No. AF7022, 1:1000, Affinity Biosciences); ERK1/2 antibody (No.4695, 1:1000, Cell Signaling Technology); p-ERK1/2 (Thr202/Tyr204) antibody (No. 4370, 1:1000, Cell Signaling Technology); AKT (No. 9272, 1:1000, Cell Signaling Technology), p-AKT (Ser473) antibody (No. 9271,1:1000, Cell Signaling Technology); cyclinD1 (AB134175, 1:20000,Abcam); β-actin (No. 10494-1-AP, 1:10000, Proteintech, Beijing, China).