Species specific secondary antibody

Poly Lactide -co-glycolide acid (PLGA) Resomer LG 857 was purchased from Boehringer Ingelheim company, 2,2,2-Trifluoroethanol (TFE) >99%, was purchased from Sigma Aldrich company. Synthetized pure Honokiol (Molecular Weight 266.334) was purchased from Sellekchem (Houston Texas). Primary antibodies against Bcl-2, Bcl-xL, HO-1 and species-specific secondary antibodies were obtained from Cell Signaling. Antibody against β-actin was purchased from Sigma Aldrich.

Cell lysis was performed using a total protein extraction buffer (Beyotime, Shanghai, China) and protein concentration was measured using a BCA Protein Assay Kit (Pierce Biotechnology). Cell lysates were separated by 6–12% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skimmed milk, the membrane was probed with one of the following primary antibodies: GRINA (1:1000, ABGENT, AP13558c), β-ACTIN (1:1000, Servicebio, GB13001–3), Bcl-2 (1:1000, Cell Signaling Technology, #4223), Bax (1:1000, Cell Signaling Technology, #5023), Cleaved-caspase3 (1:1000, Cell Signaling Technology, #9664), Cleaved-caspase7 (1:1000, Cell Signaling Technology, #8438), Cleaved-caspase9 (1:1000, Cell Signaling Technology, #9505), Cyclin D1 (1:10000, Abcam, ab134175), Cyclin E (1:1000, Abcam, ab3927), Akt (1:1000, Cell Signaling Technology, #4685), p-Akt (1:2000, Cell Signaling Technology, #4060), P70S6K (1:1000, Cell Signaling Technology, #9202), p-P70S6K (1:1000, Cell Signaling Technology, #9204), 4EBP1 (1:1000, Cell Signaling Technology, #9644), p-4EBP1 (1:1000, Cell Signaling Technology, #2855), AMPK (1:1000, Cell Signaling Technology, #2532), p-AMPK (1:1000, Cell Signaling Technology, #2535), and respective species-specific secondary antibodies (ThermoFisher Scientific). The bound secondary antibodies were detected using the Odyssey imaging system (LI-COR Biosciences, Lincoln, NE).

Honokiol (HNK) and Rapamycin (RAPA) were purchased from Selleckchem. For Western blot analysis, primary antibodies against CDK2, CDK4, CDK6, Cyclin D1, p21, Bcl-2, Bcl-xL, c-Met, phospho-c-Met, HO-1, Akt, mTOR and S6 and species-specific secondary antibodies were obtained from Cell Signaling. Antibody against β-actin was purchased from Sigma Aldrich. Anti-PD-L1, phospho-Akt, phospho-S6 and phospho-mTOR antibodies were purchased from Thermo Fisher Scientific. Recombinant human hepatocyte growth factor (HGF) was purchased from Peprotech.

Antibodies specific to Stat3 (#9132, 1:1000), p-Stat3 (#9145, 1:2000), P21 (#2947, 1:1000), β-actin (#3700, 1:1000), N-cadherin (#13116, 1:1000), E-cadherin (#3195, 1:500), and GAPDH (#2118, 1:2000) were obtained from Cell Signaling Technology. Antibodies specific to Piwil2 (ab85084, 1:1000), histone H3 (ab10799, 1:1000), H3K9ac (ab10812, 1:1000), H3K9me3 (ab71604, 1:1000), c-Myc (ab32072, 1:500), Klf4 (ab72543, 1:1000), Nanog (ab80892, 1:500), Oct4 (ab181557, 1:1000), Sox2 (ab137385, 1:500), Vimentin (ab8978, 1:500), Slug (ab27568, 1:500), Snail (ab180714, 1:500), and β-catenin (ab32572, 1:5000) were purchased from Abcam. Antibodies specific to P53 (BM0102, 1:500), Cyclin D1 (BA0770, 1:1000), and Bcl-2 (BA0412, 1:200) were obtained from Boster Technology. Protein was prepared from the cells and tissues according to the kit manual (89900, Thermo, USA) except for the addition of an acid extraction step for histones [45 (link)]. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF, IPFL00010, Merck Millipore, Germany) membranes and probed with the indicated primary antibodies. Incubation with species-specific secondary antibodies (Cell Signaling) was performed at room temperature for 1 hour. The blots were developed with chemiluminescent substrate (34080, Thermo), and autoradiography was performed with X-OMAT film (Kodak, Rochester, NY, USA).