the mutation frequency to PA 824 resistance is comparable to that of INH and has

cells were seeded onto 96 well tissue culture plates and allowed to stick for 24 hours. Cells were then produced in the presence of individual agents or combinations of drugs for 72 hours. The experiments were repeated at least 2 times and completed in triplicate. Supernatants were collected, Cabozantinib combined, and then assayed for the presence of produced VEGF utilising the Optima fluorescence plate reader. were normalized to total protein present in supernatant and compared with common curves decided using VEGF standards provided within the set. This assay effectively measures VEGF levels between 2000 pg/ ml and 9 pg/ml. Western blot analysis Total protein lysates were prepared from cells incubated in the presence of single drug, the drug combos or vehicle controls. Fleetingly, cells were gathered from plates with trypsin, rinsed with PBS, and centrifuged at 1500 g for five minutes. Cell pellets were then re-suspended in lysis buffer, Mini protease inhibitor cocktail tablets, Retroperitoneal lymph node dissection sheared using 25-gauge needles, incubated on ice for 30 minutes, and finally centrifuged at 10,000 g for 10 minutes to remove insoluble material. Protein concentrations were determined from supernatant utilising the Bradford Method and approximately 75 ug of total protein from each sample were denatured in running buffer by boiling for 10 minutes and loaded onto one hundred thousand SDS PAGE. Proteins separated by electrophoresis were used in Nitro-cellulose membrane and blocked for one hour at room temperature in Odyssey blocking buffer. Membranes were incubated at 4 C overnight in Odyssey blocking buffer containing polyclonal anti ILK, anti AKT, anti G AKT or anti Her2 antibodies. Filters were then washed 3 times for five minutes with PBS Tween and incubated with both anti rabbit or mouse IRDYE or anti rabbit Alexa 680 for one hour at room temperature and signals were detected and quantified using the Odyssey Infrared Detection AG-1478 System and associated software. and feedback variance between samples were fixed using signal intensities for negative get a handle on pixel noise and actin band intensities, respectively. Data were expressed as mean values standard deviation and parametric analysis was done utilizing an unpaired Student t test. Immunofluorescence analysis Cells grown on coverslips were rinsed with PBS, mounted using 2. Five minutes paraformaldehyde in PBS for 20 minutes at room temperature and permeabilized using 0. 5%Triton X 100 in PBS for five minutes at room temperature. Coverslips were washed three times in PBS and incubated for one hour last year BSA in PBS to prevent non specific binding, then washed three times with PBS, and then incubated with phalloidin conjugated to Texas red for 20 minutes at room temperature.