Zeb1 sc 25388

Post 72 h of culture, MIA PaCa-2R+lenti-hsamiR205 and MIA PaCa-2R+lenti-hsamiRScramble cells were lysed using RIPA buffer and protein expression was analyzed as before [49] by incubating with primary antibodies for CD44 (3578S) (Cell Signaling Technology, Inc.), ALDH1A1 (sc-166362), ZEB1 (sc-25388) and Actin (sc-1616) (Santa Cruz Biotechnology, Inc.). Post-incubation with primary antibody for 16 h at 4 °C, HRP-conjugated secondary antibodies (sc-2020, sc-2030 and sc-2031) (Santa Cruz Biotechnology, Inc.) were applied for 1 h in phosphate buffered saline with Tween^® 20 (PBST) at RT. Target proteins were detected by ImmunoCruz Western blotting luminol reagent (Santa Cruz Biotechnology, Inc.).

Protein preparation, immunoblotting, and immunohistochemistry were performed as described (19 (link)) (20 (link)). For immunofluorescence, cells grown on coverslips were fixed with Carnoys solution (6:3:1 ethanol:chloroform:acetic acid), and biotinylated secondary anti-mouse or anti-rabbit antibody (Vector Laboratories; Burlingame, CA) binding was detected with fluorescein isothiocyanate (FITC)-conjugated avidin (Vector Laboratories) or AlexaFluor 594-conjugated avidin (Life Technologies Corp., Carlsbad, CA). Coverslips were mounted on slides with mounting medium containing DAPI (Vector Laboratories). Antibodies against human PTK6 (G6, sc-166171), E-Cadherin (sc-7870), and ZEB1 (sc-25388) were purchased from Santa Cruz Biotechnology. Antibodies against N-Cadherin (13116), ZO-1 (8193), Vimentin (5741), Claudin-1 (13255), and GAPDH (2118) were purchased from Cell Signaling Technology, and β-Actin (A5411) from Sigma-Aldrich. Antibodies against inactive PTK6 (PY447; ab138368), and Ki67 (ab16667) were purchased from Abcam (Cambridge, MA). Antibody against active PTK6 (PY342; 09-144) and was purchased from EMD Millipore (Temecula, CA). Densitometry analysis was done using ImageJ software (NIH) and values were normalized to loading control.