Anti actin c 2

The primary antibodies used anti-E-cadherin (Clone 36; BD Transduction Laboratories, San Jose, CA, USA), anti-ZEB1 (H-102; Santa Cruz), anti-actin (C-2; Santa Cruz), anti-FLASH (M-300; Santa Cruz), anti-caspase-3 (8G10; Cell Signaling Technology Inc, Danvers, MA, USA), anti-cleaved PARP-1 (D214; Cell Signaling), anti-lamin A/C (4C11; Cell Signaling), anti-ubiquitin (P4D1; Cell Signaling), anti-phospho-SMAD2 (D27F4; Cell Signaling), anti-SMAD2/3 (D7G7; Cell Signaling), anti-SNAIL (C15D3; Cell Signaling) and anti-SLUG (C19G7; Cell Signaling) were obtained commercially. Secondary antibodies horseradish peroxidase-conjugated anti-mouse and anti-rabbit (1:5000) were from Jackson Laboratories (West Grove, PA, USA). Gemcitabine was purchased from Tocris (Bristol, UK). MG132 and TGFβ were purchased from Cell Signaling.

MDCK cells in 35 mm dishes were infected with MFPT^r virus or the WT virus at 1 PFU/cell or mock infected with PBS. At 0, 5, 10 and 15 hours post-infection, the infected cells were washed 3 times with PBS and stored at -80°C. Using cell lysis buffer (0.05 M Tris-HCl pH 7.0, 0.15 M NaCl, 1% SDS, 1% Triton X-100), the cell extracts were prepared and subjected to 10% SDS-PAGE. The resulting gels were blotted on PVDF membranes and soaked with 2% BSA in TBS for blocking treatment. Blotted proteins were immuno-reacted with rabbit anti-Akt antibody (Cell Signaling Technology, MA, USA), anti-phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling Technology), anti-cleaved caspase 3 (Cell Signaling Technology), anti-PARP-1 (Santa Cruz Biotechnology, TX, USA), anti-Actin(c-2) (Santa Cruz Biotechnology), anti-Influenza A m1 (Santa Cruz Biotechnology), or anti-Influenza A ns1 (Santa Cruz Biotechnology) antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology) were used as secondary antibodies. Bands of these proteins were detected using TMB Stabilized Substrate for HRP (Promega KK, Tokyo, Japan). Semi-quantifying the expression levels of these proteins were performed using an image analyzing software, ImageJ [12 (link)] and that of actin for data normalization.

Immunoblot analysis and Immunofluorescence staining were carried out with the following antibodies: anti-F4/80 (AbD Serotec), anti–ERα D6R2W (13258 Cell Signaling), and ab3575 (Abcam, Cambridge, UK), anti-Actin C-2 (sc-8432 Santa Cruz, Santa Cruz, CA, USA), and anti-GAPDH (RPCA Encor, Gainesville, FL, USA).