Rabbit anti e cadherin

Total protein was extracted from the MPC5 cells using RIPA lysis buffer (Beyotime, Jiangsu, China) following the manufacturer's instructions. Western blot analysis was performed as previously described (25 (link)). The antibodies used were the following: rabbit anti-desmin (1:1,000; sc-14026), rabbit anti-snail (1:1,000; sc-28199) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-nephrin (1:2,000; ab136894; Abcam, Cambridge, MA, USA), rabbit anti-Wilms tumor 1 (WT1; 1:500; sc-192), rabbit anti-E-cadherin (1:1,000l; sc-7870) (both from Santa Cruz Biotechnology, Inc.), rabbit anti-GSK3β (1:2,000; ab18893; Abcam), rabbit anti-β-catenin [activated and unphosphorylated, 1:2,000; Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands], mouse anti-β-tubulin (1:3,000; sc-80011), goat anti-rabbit IgG-HRP (1:3,000; sc-2004) and goat anti-mouse IgG-HRP (1:3,000; sc-2005) (all from Santa Cruz Biotechnology, Inc.). For the quantitative analysis of the western blots, the protein band intensities were quantified using Image J software and β-actin was used for normalization.

Protein extracts were prepared as previously published by our laboratory; cells were homogenized on ice in RIPA buffer (50 mM Tris, pH 8.0 containing 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, and 1% NP40) containing protease inhibitors. Protein concentrations were measured using the Bradford method. Samples were mixed with loading buffer containing β-mercaptoethanol and boiled for 5 min. One-hundred micrograms of each sample were then separated in SDS-PAGE mini gels (BioRad) and transferred to PVDF membranes (Amersham Biosciences, Uppsala, Sweden). The membranes were incubated for 1h at room temperature in blocking buffer (5% fat free milk, 0.1% Tween-20 in PBS). Primary antibodies were used at a 1/200–1/2000 dilution in blocking buffer and were incubated at 4 °C overnight. After washing with PBST, membranes were incubated with secondary antibodies at a 1/1000 dilution for 1 h at room temperature. Signals were detected with an enhanced chemiluminescence kit (ECL, Amersham Biosciences). Primary antibodies: rabbit anti-ER alpha, rabbit anti-E-cadherin, rabbit PR (Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibody: donkey-anti-rabbit HRP.

Cells in 6-well tissue culture plates were lysed in 0.5 mL of lysis buffer (PBS containing 1% Triton X-100 and 1 mM PMSF) at 4°C for 10 min. Equal quantities of protein were subjected to SDS–PAGE under reducing conditions. Following a transfer to immobilon-P transfer membrane, successive incubations with a primary antibody and a horseradish peroxidase-conjugated secondary antibody were carried out for 60–120 min at room temperature. The immunoreactive proteins were then detected using the ECL system. Films showing immunoreactive bands were scanned using an HP Scanjet 5590 scanner (HP, Pal Alto, CA, USA). The antibodies used for Western blotting were: mouse anti-HA (Roche, Basel, Switzerland), monoclonal mouse anti-FLAG M2 (Sigma, St. Louis, Mo, USA), anti-SPOP (Abcam, Cambridge, United Kingdom), monoclonal rabbit anti–ITCH (Cell Signaling, Danvers, MA, USA), rabbit anti-E-cadherin (Santa Cruz, Dallas, TX, USA), mouse anti-FLAG-HRP (Sigma), mouse anti-HA-HRP (Roche), anti-rabbit IgG-HRP (Sigma), and anti-mouse IgG-HRP (Sigma).

Cells were resuspended in lysis buffer (100 mM NaCl, 15 mM MgCl₂, 2 mM EDTA, 20 mM Hepes, 10% Glycerol, 1 mM dithiothreitol, 2 mM Na-ortovanadate, Protease-Inhibitor Cocktail (Sigma-Aldrich) and 1% Triton X-100). After10 min of incubation in ice, extracts were centrifuged for 10 min at 12 000 rpm at 4 °C, supernatants were resuspended in SDS-page sample buffer and boiled for 5 min. Western blot analysis was performed as previously described.^{31 (link)} The following primary antibodies (overnight at4 °C) were used: rabbit anti-Actin (1 : 1000, Sigma-Aldrich), mouse anti-GAPDH (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-E-cadherin (1 : 1000, Santa Cruz Biotechnology), mouse anti-Vimentin (1 : 1000, Santa Cruz Biotechnology), rabbit anti-ZEB1 (1 : 1000, Sigma-Aldrich), rabbit anti-eiF4E (1 : 1000, Cell Signalling Technology, Danvers, MA, USA), rabbit 4EBP1 (1 : 1000, Cell Signalling Technology), rabbit anti PARP-1 (1 : 1000, Cell Signalling Technology), rabbit anti eiF4A (1 : 1000, Abcam, UK), rabbit anti-eIF4G (1 : 1000, Cell Signalling Technology). Secondary anti-mouse or anti-rabbit IgGs conjugated to horseradish peroxidase (Amersham, UK) were incubated for 1 h at RT (1 : 10 000). Immunostained bands were detected by chemiluminescence method (Santa Cruz Biotechnology).

LPS and EGF were purchased from Sigma-Aldrich, n-Octyl-D-glucoside was purchased from Santa Cruz Biotechnology. Primary antibodies were: mouse monoclonals Eno 276/3 and 72/1 against alpha-enolase^{19 (link)}; mouse monoclonal cmHsp70.1 against membrane Hsp70^{24 (link)}; goat anti-alpha-enolase, rabbit anti-E-cadherin, mouse anti-Hsp90, rabbit anti-Hsp70, goat anti-GAPDH, rabbit and mouse anti-EGFR, mouse anti-TFR, rabbit anti-uPAR, rabbit anti-Vimentin, goat anti-Lamin B (Santa Cruz Biotechnology); rabbit anti-phospho-Akt, mouse anti-Akt, rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technologies); mouse anti-ERK1/2 (Proteintech Group), mouse anti-β-actin (Sigma-Aldrich); rabbit anti-TLR4 (Novus Biologicals); rabbit anti-Caveolin-1 (Upstate Biotechnology); mouse anti-Annexin-A2 (BD Biosciences). Alexa Fluor-conjugated secondary antibodies were purchased from ThermoFisher Scientific Inc.

Cell lysates were made with Laemmli buffer (for Western blotting) or with TNT-buffer (pulldown experiments; 150 mM NaCl, 50 mM Tris, 1 mM MgCl₂, 1 mM CaCl₂, 1% Triton X-100, pH 7.5 + protease inhibitor cocktail). SDS-PAGE and Western blotting were performed according to standard procedures. The following primary antibodies were used: mouse anti-Pals1 (1:500, Santa Cruz #365411), rabbit anti-Pals1 (1:1000, Proteintech 17710-1AP), mouse anti-ß-Actin (1:500, Santa Cruz #47778), mouse anti-Rac (1:500, Santa Cruz #514583), rabbit anti-α-Actinin 4 (1:1000, Cell Signaling #6487), mouse anti-α-Actinin 4 (1:2000, Santa Cruz #17829), mouse anti-Arf6 (1:500, Santa Cruz #7971), rabbit anti-E-Cadherin (1:1000, Santa Cruz #7870), mouse anti-SMAP1 (1:1000, Abnova H00060682-B01P).