Monoclonal tubulin antibody

Total-AKT, phospho-AKT (Thr308), total-ERK1/2, phospho ERK1/2, total PDK1, phospho-PDK1, Phospho-IGF-I Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) (19H7), total IGFR, PTEN and BRaf (55C6) were purchased from Cell Signaling Technology (Danvers, MA); Anti-p16INK4a antibody was purchased from Neo Markers (Fermont, CA); anti-mutated BRaf (V600E) antibody was purchased from New East Biosciences (Malvern, PA); anti-Grm1 antibody was purchased from R&D Systems (Minneapolis, MN); anti-p15 was from Santa Cruz; anti-β actin was from ThermoFisher; monoclonal -Tubulin antibody was obtained from Sigma (St. Louis, MO); anti- rabbit secondary was purchased from Merck ; anti-mouse secondary was purchased from Sigma (St. Louis, MO). Anti-sheep secondary was purchased from R&D Systems (Minneapolis, MN).

HeLa cells (ATCC^® CCL-2™, American Type Culture Collection) were grown in Dulbecco’s modified eagle medium supplemented with 10% fetal calf serum and 100 μg/ml kanamycin in a humidified incubator at 37 °C with 5% CO₂. Cells were grown on 12-mm-diameter glass coverslips for microscopic analysis. For the evaluation of the BiFC signal, HeLa cells were transfected with different mVenus constructs of TPPP/p25 (0.3 μg of each plasmid) using Turbofect (Invitrogen) transfection reagent according to the manufacture’s protocol. Monoclonal tubulin antibody (Sigma T9026) and an Alexa 546 conjugated mouse secondary antibody (Thermo Fisher Scientific A11030) were used for the detection of the tubulin signal. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images of the mounted samples were acquired on a Leica DM IL 500 microscope equipped with Leica DFC 395 FX camera and HBO 100w lamp. The equipment software was Leica Application Suite 4.4.0. Chroma UV filter set (No. C40888), Chroma 41028 HQ NB GFP filter set (No. C21116) and Leica filter N2.1 (No. 513832) was used for DAPI, BiFC and Alexa 546 signal acquisition, respectively, using a HCX FL Fluotar 40x/0.75 (dry) objective.