Anti p akt akt

MCF-10A, MCF-10A-ras, MDA-MB-453, MDA-MB-468, SKBR3, MCF-7, T47D, and CCD-1068sk cells were originally obtained from American Type Culture Collection, and the BT474 cell line was obtained from Korean Cell Line Bank. The cells were cultured according to the standard procedure and maintained at 37 °C in a humidified atmosphere composed of 5% CO₂/95% air. GREM1 antibody was purchased from Abcam and recombinant human GREM1 was obtained from R&D systems. Recombinant human EGF, anti-Flag antibody and cell linker kits (PKH26 and PKH67) were purchased from Sigma-Aldrich. Anti-ERRα, anti-p-EGFR/EGFR, anti-p-Akt/Akt, anti-p-ERK/ERK antibodies and erlotinib were obtained from Cell Signalling Technology. Expression plasmids of GREM1, ERRα, Flag-only, Flag-EGFR, and Flag-BMP2 were purchased from Sino Biological Inc. Fc-IgG1 and Fc-GREM1 were provided by ACROBiosystems. The lentiviral GREM1 clone was obtained from Genecopoeia. 3xERRE-luciferase, pcDNA4-myc-PGC-1α, EGFR-WT, EGFR-ECD and EGFR-ICD were provided by Addgene. XCT790, LY294002 and U0126 were purchased from Tocris.

MDA-MB-453 and Hs578T cells were originally obtained from American Type Culture Collection. MDA-MB-453 cells were cultured in RPMI (Corning, NY, U.S.A.) containing 10% fetal bovine serum (FBS, Gibco, NY, U.S.A.) and 100 units/ml penicillin/streptomycin (Corning). Hs578T cells were cultured in DMEM (Corning, NY, U.S.A.) containing 10% FBS and 100 units/ml penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂/ 95% air. DHA (purity > 98%) was purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). Rabbit polyclonal GREM1 antibody was purchased from Abcam (Cambridge, U.K. Cat#. 140010). Recombinant human GREM1 was obtained from R&D systems (MN, U.S.A., Cat#. 5190-GR). Anti-N-cadherin (Cat#. 13116), anti-vimentin (Cat#. 5741), anti-Slug (Cat#. 9585), anti-pERK/ERK (Cat#. 8201), anti-pAkt/Akt (Cat#. 8200), and β-actin (Cat#. 4967) were obtained from Cell Signaling Technology (MA, U.S.A.). Recombinant human TGF-β (Cat #. 10804-HNAC) was purchased from Sino Biological Inc. (Beijing, China). U0126 (Cat#. 1144) was purchased from Tocris Bioscience (MN, U.S.A.).

The lung tissues were homogenized using a homogenizer with RIPA buffer containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor PhosSTOP (Roche). Total protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% nonfat milk and then processed with primary antibodies: anti-p-Akt/Akt (1:1000), anti-NLRP3 (1:1000) and anti-ASC (1:1000) (cell signaling technology Inc., Beverly, MA, USA), anti-caspase-1 (1:500), anti-IL-18 (1:500) and anti-IL-1β (1:500) (Santa Cruz Biotechnology, CA, USA), and anti-GAPDH (1:2000) (KANGCHEN Biotech, Shanghai, China). All of the membranes were subsequently incubated with HRP-conjugated anti-rabbit IgG (Promega, Madison, WI, USA), polyclonal rabbit anti-mouse IgG (Dako, Copenhagen, Denmark), and all of the blots were detected via enhanced chemiluminescence (ECL; Thermo Scientific). For quantitative analysis, the intensity of protein bands was determined using Image J 1.38 × software (NIH, Bethesda, MD, USA).