Paxillin antibody

BxPC3 cells (1 × 10⁴ cells) were seeded in μ-Slide 8 well-ibiTreat, tissue culture-treated (IBIDI 80826) and incubated overnight. Cells were fixed with 2 % paraformaldehyde in PBS for 20 min, then permeabilized with 0.1% Triton X-100 for 20 min and blocked with BSA and 1% FBS for 10 min. Cells were stained with Paxillin antibody for 2 h (1:200 BD Transduction Laboratories 612405), then incubated with a secondary antibody for 1 h (1:100 Goat anti-Mouse Alexa Fluor 488 A-11029). Antifade Mounting Medium with DAPI (Vector laboratory H-1200) was used to stain the nuclei and mount the samples. Images were acquired using a spinning disk confocal microscope (Nikon) with 60× and 100× objectives. The fields of view were randomly chosen. Image analysis was performed using NIS Elements version 4.13 imaging software.

MeWo and B16F10 cell lines were purchased from the American Tissue Culture Collection. The following antibodies were from Cell Signaling Technology: phospho AKT (CAT. #: 4060), AKT (CAT. #: 2920), phospho ERK1/2 (CAT. #: 9101), ERK1/2 (CAT. #: 4696), phospho Paxillin (CAT. # 2541), phospho S6K (CAT. #: 9205), S6K (CAT. #: 9202), PCNA (CAT. #:2586), PARP (CAT. # 9542), cleaved PARP (CAT. # 9541), phosphor Src (CAT. #: 2105), Src (CAT. #: 2108), horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse, CAT. #: 7076, anti-rabbit, CAT. #: 7074). The following antibodies were from Santa Cruz Biotechnology: GAPDH (CAT. #: sc-59541), SHP2 (CAT. #: sc-280), HA (CAT. #: sc-7392). Paxillin antibody (CAT. #: 610569) was from BD Transduction Laboratories. Ki67 antibody was from Thermo Fisher Scientific (CAT. # RM-9106-S0). HGF, MTT (3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma and prepared according to the manufacture's instruction. Eagle's Minimum Essential Medium (EMEM) and Dulbecco's Modified Eagle's Medium (DMEM) were from Corning, fetal bovine serum was from HyClone. SuperSignal West Dura Luminol/Enhancer solution was from Thermo Fisher Scientific. Lipofectamine® RNAiMAX and Lipofectamine® 2000 Transfection reagent was from Invitrogen Life Technologies.