Rabbit anti p akt ser473

Mouse anti-HSPB8 (MAB4987) was from R&D System-Biotechne (Minneapolis, MN, USA). Rabbit anti-HSPB8 (PA5-76780) and rabbit anti-SQSTM1/p62 (PA5-20839) were from ThermoFisher Scientific (Waltham, MA, USA). Rabbit anti-E-cadherin (#3195), rabbit anti-N-cadherin (#13116), rabbit anti-vimentin (#5741), rabbit anti-ERK1/2 (#4695), rabbit anti-pERK1/2 (#4370), rabbit anti-cyclin D (#2926), rabbit anti-CDK4 (#12790), rabbit anti-Akt1 (#2938), rabbit anti-pAkt (Ser473) (#9271), rabbit anti-mTOR (#2983), rabbit anti-p-mTOR (Ser 2448) (#5536), rabbit anti-ATG5 (#12994) were from Cell Signaling (Danvers, MA, USA). Rabbit anti-BRAFV600E (RM8) was from RevMAb Biosciences (San Francisco, CA, USA). Mouse anti-pan-RAS (C-4) (sc-166691) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-LC3 (L8918) and mouse anti-alpha-tubulin (T6199) were from Sigma–Aldrich (St. Louis, MO, USA). Rabbit and mouse Horseradish-peroxidase-conjugated secondary antibody were from Cell Signaling. 3-Methyladenine (3-MA) (S2767) was from Selleckchem (Munich, Germany). Chloroquine (CQ) (C6628) was from Sigma–Aldrich.

Western blotting of proteins was performed as described in published protocols (18 (link)). The cell lysates or immunoprecipitated proteins were analyzed by SDS-PAGE. After completion of electrophoresis, the proteins were transferred to a polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane, followed by blocking at 4°C for a minimum of 1 h. The blots were probed overnight at 4°C with the primary antibodies. The antibodies used were as follows: mouse anti-Ago2 (Abnova), 1:1,000; rabbit anti-DICER1 (Bethyl), 1:8,000; rat anti-HA (Roche), 1:1,000; horseradish peroxidase (HRP)-conjugated anti-β-Actin (Sigma), 1:10000; rabbit anti-TRBP2 (Cell Signalling), 1:1000; rabbit anti-Drosha (Bethyl), 1:8,000; rabbit anti-P-p38 (Cell Signaling), 1:1,000; rabbit anti-P-ERK1/2 (Cell Signaling), 1:1,000; mouse anti-HSP70 (Santa Cruz Biotechnology), 1:1,000; rabbit anti-MSK1 (Cell Signaling), 1:1,000; mouse anti-HSP70 (Santa Cruz Biotechnology), 1:1,000; rabbit anti-cleaved PARP (Cell Signaling), 1:1,000; rabbit anti-cleaved caspase 9 (Cell Signaling), 1:1,000; and rabbit anti-P-Akt (Ser-473) (Cell Signaling), 1:1,000. Visualization of all Western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software (UVP) V6.80. ImageJ software was used for densitometric analysis of blots for the relative quantification of bands.

For Western blotting, equivalent amounts of sample protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Macherey Nagel, Düren, Germany). After blocking with 5% BSA in TBS-T (20 mM Tris pH 7.6, 150 mM NaCl and 0.02% Tween-20), the membrane was immunoblotted with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit anti-LRRC8A (1 μg/mL, [4 (link)] kindly supplied by T. J. Jentsch); rabbit anti-p-ULK (1:1000, Cell Signaling, Frankfurt am Main, Germany; #14202); rabbit anti-ULK (1:1000, cell signaling, #8054); rabbit anti-p-Akt^Ser473 (1:1000, Cell Signaling; #4060); rabbit anti-Akt^pan (1:1000, Cell Signaling; #4685); and rabbit anti-GAPDH (1:2500, Cell Signaling; #2118) antibodies. The corresponding peroxidase-labeled secondary antibody (1:10,000, Jackson ImmunoResearch, Ely, UK) was used. Signals were detected using an enhanced chemiluminescence reagent (HRP juice; PJK GmbH, Kleinblittersdorf, Germany) and a ChemiSmart5000 digital imaging system (Vilber-Lourmat, Collégien, France). Densitometrical quantification was performed with the Fiji software [64 (link)].

Fresh-frozen tissues were lysed with RIPA (Beyotime, China) containing a protease inhibitor mixture (protease inhibitors; phosphatase inhibitors; PMSF; KangChen, Shanghai, China) on ice for 30 minutes. The concentration of protein was measured by a BCA protein assay kit (Pierce Biotechnology). Proteins were separated on 10% SDS-polyacrylamide gels and transferred on to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Then, the membranes were blocked with 5% nonfat milk and incubated with the primary antibodies at 4°C overnight. After that, the membranes were washed with TBST and incubated with HRP-labeled goat anti-rabbit antibody (1:5000, WeiAo, Shanghai, China) for 1 hour. Finally, the immunoreactive signals were detected using the ECL detection system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). Sources of antibodies included rabbit anti-GPR116 (1:200, Proteintech Group, USA), rabbit anti-N-Cadherin (1:1000, Cell Signaling Technology, USA), rabbit anti-E-Cadherin (1:1000, Cell Signaling Technology, USA), rabbit anti-Snail (1:1000, Cell Signaling Technology, USA), rabbit anti-p-AKT (Ser473) (1:2000, Cell Signaling Technology, USA) and rabbit anti-p-ERK1/2 (Thr202/Tyr204) (1:2000, Cell Signaling Technology, USA), GAPDH (1:3000, KangChen, Shanghai, China).