Gapdh sc 365062

Samples were resolved by SDS-PAGE (10–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated. Primary antibodies were used to detect Bcl-x_L (54H6, 1:1,000 dilution, Cell Signaling; 2H12, 1:500 dilution, Invitrogen), COX IV (3E11, 1:20,000 dilution, Cell Signaling), Drp1 (D6C7, 1:1,000 dilution, Cell Signaling), Flag (66008-3-Ig, 1:1,000 dilution, Proteintech), GAPDH (sc-365062, 1:1,000 dilution, Santa Cruz biotechnology), GFP (sc-8334, 1:1,000 dilution, Santa Cruz biotechnology), HA (51064-2-AP, 1:1,000 dilution, Proteintech), Mff (17090-1-AP, 1:2,000 dilution, Proteintech), PARP (46D11; 1:1,000 dilution, Cell Signaling), RFP (3F5, 1:1,000 dilution, ChromoTek), SENP3 (D20A10, 1:10,000 dilution, Cell Signaling), and β-actin (A2228, 1:20,000 dilution, Sigma). Immune complexes were detected either using HRP-conjugated secondary antibodies (Sigma) or an HRP-conjugated VeriBlot secondary antibody (ab131366, Abcam; for immunoblotting involving IP samples) followed by enhanced chemiluminescence (GE Healthcare Amersham), or using fluorescent secondary antibodies (Thermo Fisher Scientific) by a LI-COR imaging system. Each immunoblot presented is representative of at least three independent experiments carried out using different cell populations.

DMEM, FBS, goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488, Fura-2 AM and all RT-qPCR reagents were obtained from Invitrogen (Carlsbad, CA). Neurotensin, insulin and phalloidin-TRITC were obtained from Sigma Chemical (St. Louis, MO). The geranylgeranyl transferase I (GGTase I) inhibitor GGTI 298, PI 3-Kinase inhibitor A66 and the MEK inhibitor PD0325901 were from R&D Systems (Minneapolis, MN). The dual PI3K/mTOR inhibitor NPV-BEZ235 was purchasedfrom Selleck Chemicals (Houston, TX). Primary antibodies used were as follows: YAP (H-9, sc-271134 and 63.1, sc-101199, final dilution 1:200 for immunofluorescence), tubulin (sc-5274; final dilution 1:400), actin (sc-47778; final dilution 1:400) and GAPDH (sc-365062; final dilution 1:400) (Santa Cruz Biotechnology); phospho-YAP Ser127 (D9W2I, 13008; final dilution 1:1000), YAP (15028; final dilution 1:1000 for western blots), phospho-p70 S6 KinaseThr389) (9205; final dilution 1:1000) and phospho-S6 Ribosomal Protein Ser240/244 (5364; final dilution 1:1000), phospho LATS Thr1079 (8654; final dilution 1:1000) and LATS2 (5888; final dilution 1:1000) were from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase–conjugated anti-rabbit IgG and anti-mouse IgG were from GE Healthcare Bio-Sciences Corp (Piscataway, NJ). All other reagents were of the highest grade available.

The IHC staining was performed based on the protocol described in previous study [22 (link)]. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-365062), α-synuclein (sc-12767) and MAP-2 (sc-74422) antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Protein from the cell extracts was resolved by electrophoresis and transferred to poly vinylidene difluoride membrane (PVDF). After blocking with 5 % non-fat milk in Tris-buffered saline, membranes were incubated with specific antibodies at 4 °C overnight. The membrane was then incubated with horseradish peroxidase labeled secondary antibodies. Proteins were visualized with the Super Signal West Femto Maximum sensitivity substrate kit (Thermo Scientific, Logan, UT). Western blots were quantified using the software Image Pro Plus version 6. The antibodies to ANG-2 (SC-7015) and GAPDH (SC-365062) were purchased from Santa Cruz Biotechnology (USA). The antibodies to MUC4/Y (MUC4) (#ab60720), VEGF-A (#ab51745), CD31 (#ab28364) and Hes-1 (#ab71559) were bought from Abcam (USA). The selected monoclonal antibody (#ab60720, Abcam, UK) is specifically directed against amino acids 79–189 of human MUC4, which are included in the protein expressed by the MUC4/Y target gene, including the AMOP, NIDO, VWD, and the EGF-like domains. The antibodies to NOTCH3 (#5276), and MMP-9 (#13667) were from Cell Signalling Technology (USA). Pre-stained markers (Thermo Scientific, USA) were used as internal molecular weight standards. Each blot was independently repeated three times.