Anti vimentin antibody

Three kidney cancer tissues and corresponding histologically proven non-malignant tissues adjacent to the tumor were obtained after surgery. All the tumors were of Grade 3. Portions of the tissue were immediately sent to the histopathology laboratory for histological diagnosis. Other portions were placed into a transport medium and disaggregated immediately as described previously by Valente et al. [18 (link)]. The cultured cells exhibited the characteristic features of epithelial cells (i.e., positive immunocytochemical staining for cytokeratin 19). Contamination from fibroblasts was quantified using an anti-vimentin antibody (Sigma, St. Louis, MO, USA). Their expression was lower than 10%. In all experiments, to obtain enough cells to perform a basic growth assay and ERK1/2 activation, and to avoid variations in the characteristic features of the cells, normal and corresponding tumor kidney epithelial cells were exclusively used in the primary culture at passage 2.

Cytosolic and nuclear extracts were obtained using an Active Motif nuclear extraction kit (Active Motif) according to the manufacturer’s instructions.
Cytosolic and nuclear extracts were separated by sodium dodecyl sulfate–​polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane sheets (Immobilon-P, Millipore) and probed with the required antibody diluted in 0.1% PBS-Tween with 5% nonfat dry milk. After 1 hr of incubation, the membranes were washed with 0.1% PBS-Tween and then were incubated for 1 hr with peroxidase-conjugated sheep anti-mouse or sheep anti-rabbit IgG antibody (Amersham International) diluted 1:3,000 in 0.1% PBS-Tween with 5% nonfat dry milk. The membranes were washed again with 0.1% PBS-Tween, and proteins were detected by enhanced chemiluminescence (Perkin Elmer).
Anti-E-cadherin, β-catenin, tubulin, SNAIL-1, and TATA-binding protein (TBP) antibodies were all provided by Santa Cruz Biotechnology, Inc. Tubulin and TBP were used as loading controls for the cytosol and the nucleus, respectively. The anti-vimentin antibody was provided by Sigma Chemical Co. The anti-α-SMA antibody was from GeneTex. The anti-Smad2 and p-Smad2 antibodies were from Abcam.

Plasmids for lentiviral vector construction were obtained from Addgene (Cambridge, MA). PCR primers were synthesized by Integrated DNA Technologies (Coralville, Iowa). The qPCR lentivirus titration kit was purchased from Applied Biological Material (Atlanta, GA). The PBS, Triton^TMX-100, voltage-sensitive fluorescent dye RH-237, and Alexa Fluor 633 secondary antibody were purchased from Life Technologies (Carlsbad, CA). The anti-vimentin antibody, the anti-sarcomeric-α-actinin antibody, and all other reagents were from Sigma-Aldrich (St. Louis, MO).
Data were analyzed using OriginLab software. Comparisons between groups were performed using unpaired 2-tailed Student’s t-test (Figs 1, S3 and S7 and Table 1) or One-Way or Two-Way ANOVA test (Figs 2, S2 and S4). Data were considered significantly different at p < 0.05. Results are presented as bar (mean ± SEM) or box chart.