Phospho s6 ribosomal protein ser240 244

GFP was examined either directly by fluorescence microscopy or by immunostaining on frozen sections using a chicken polyclonal GFP antibody (Abcam, Cambridge, MA). CD31 immunostaining was performed on frozen sections using a rat CD31 antibody (BD Biosciences, San Jose, CA). To detect P-S6, paraffin sections were de-paraffinized, treated with trypsin for 10 minutes, and blocked with 10% sheep serum before being incubated with a rabbit polyclonal antibody against Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signaling Technology, Danvers, MA).

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 pieces of ear skin were lysed with cold radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Subsequently, the collected protein was subjected to sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and blotted using nitrocellulose paper (Amershan). The membrane was probed with antibodies against Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8, Cell Signaling Technology, Danvers, MA, USA) and Phospho-Akt (Ser473) (193H12, Cell Signaling Technology). GAPDH (D16H11, Cell Signaling Technology) was considered a loading control.

Serial 3-μm-thick paraffin sections from original tumors and PDXs were processed according to standardized automated procedures (Ventana Medical Systems) and then immunostained with the following antibodies: CD99 (Ventana, mouse monoclonal antibody O13, prediluted), ETV4 (Santa Cruz Biotechnology, clone PEA3, 1/20), or buffer alone (negative control); p-AKT (Ser473; 736E11, rabbit, #3787, 1:50, Cell Signaling Technology, RRID:AB_331170); phospho-mTOR (Ser2448; polyclonal, rabbit, #2971S, 1:50, Cell Signaling Technology, RRID:AB_330970); phospho-S6 ribosomal protein (Ser240/244; rabbit, #2215, 1:30, Cell Signaling Technology, RRID:AB_2238583). For morphologic analyses, the slides were stained with hematoxylin/eosin.

The primary antibodies include GAPDH (goat pAb; SAB2500450, Sigma), βTub (rabbit pAb; ab6046, Abcam), α-actinin (rabbit mAb; 6487, Cell Signaling), HER2-3B5 (mouse mAb; ab16901, Abcam), HER2-2G11 (mouse mAb; AHO0918, Thermofisher Scientific), ERK1/2 (rabbit mAb; 4695, Cell Signaling), phospho-ERK1/2 (Thr202/Tyr204) (rabbit mAb; 4370, Cell Signaling), mTOR (rabbit mAb; 2983, Cell Signaling), panCK (rabbit pAb; Z0622, Dako), S6-ribosomal protein (mouse mAb; 2317, Cell Signaling), phospho-S6-ribosomal protein (Ser240/244) (rabbit mAb; 5364, Cell Signaling). The secondary antibodies include donkey anti-mouse IgG (H + L) (Alexa Fluor 488 conjugate, A21202, Thermofisher Scientific), donkey anti-goat IgG (H + L) (Alexa Fluor 555 conjugate, A21432, Thermofisher Scientific), and donkey anti-rabbit IgG (H + L) (Alexa Fluor 647 conjugate, A31573, Thermofisher Scientific).