Amersham ecl prime detection reagent

Immunoblot analysis was performed as described previously [6 (link),16 (link)]. In brief, cells were washed once with ice-cold PBS to remove serum proteins and lysed with 1× PhosphoSafe lysis buffer (Merck Millipore). Following clearance of the lysates by centrifugation, their total protein content was determined with the DC Protein Assay (BioRad). Proteins were fractionated by polyacrylamide gel electrophoresis on mini-PROTEAN TGX any-kD precast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies overnight at 4 °C. After washing and incubation with HRP-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS+ System with Image Lab Software (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to those for the housekeeping gene GAPDH.

Our Western blotting procedure was described in detail earlier [16 (link),17 (link),43 (link)]. Total protein concentrations were determined with the DC Protein Assay (BioRad). Proteins were fractionated by PAGE on mini-PROTEAN TGX any-kD precast gels and blotted onto PVDF membranes. The primary antibodies included anti-HSP90 (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-13119), anti-Rac1b (Merck Millipore, Darmstadt, Germany, #09-271), anti-E-cadherin and anti-Cip1/WAF1 (BD Transduction Laboratories, Heidelberg, Germany, #610181 and #610233, respectively), anti-Snail and anti-phospho-ERK1/2 (Cell Signaling Technology, Frankfurt/Main, Germany, #4719 and #4370, respectively), anti-ERK1/2 (R&D Systems, Wiesbaden, Germany, #AF1576), anti-GAPDH (14C10, Cell Signaling Technology, #2118), and anti-β-actin (Sigma). Incubation with HRP-linked secondary antibodies (Cell Signaling Technology, anti-rabbit, #7074, and anti-mouse, #7076) was followed by chemoluminescent detection of proteins on a ChemiDoc XRS+ System with Image Lab Software (BioRad) using Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to bands for the housekeeping genes GAPDH or HSP90.

Cell lysis and immunoblotting were essentially performed as described previously [8 (link),9 (link),10 (link)] with minor modifications. Proteins were fractionated by polyacrylamide gel electrophoresis on TGX Stain-Free FastCast gels (BioRad, Munich, Germany). Following blotting and antibody treatment, chemoluminescent detection of proteins was carried with a ChemiDoc XRS+ System with Image Lab Software (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to either the total amount of protein in the same lane (when using the TGX Stain-Free FastCast gels), or to bands for the housekeeping gene GAPDH. Significant differences (p < 0.05)-calculated with the unpaired two-tailed Student’s t test–denoted by bars above the respective cell lines. The antibodies used were Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), E-cadherin (#610181) and Rac1 (#610650) (BD Transduction Laboratories, Heidelberg, Germany), GAPDH (14C10, #2118, Cell Signaling Technology, Frankfurt am Main, Germany), Vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), and HA (clone 12CA5, Roche Diagnostics, Mannheim, Germany).

Western blots were performed as previously described (Chia et al., 2017 (link)). Protein extracts were prepared using the trichloroacetic acid (TCA) extraction protocol. Cells were collected by centrifugation and re-suspended in cold 5% w/v TCA for at least 10 min. Samples were washed with acetone, then completely air-dried. Cells were resuspended with protein breakage buffer (50 mM Tris (pH 7.5), 1 mM EDTA, 2.75 mM dithiothreitol (DTT)) and disrupted using 0.5 mm glass beads and a Mini Beadbeater (Biospec). Two volumes of protein extract were mixed with 1 volume of SDS-PAGE sample buffer (187.5 mM Tris (pH 6.8), 6.0% v/v β-mercaptoethanol, 30% v/v glycerol, 9.0% v/v SDS, 0.05% w/v Bromophenol blue) and denatured at 95°C for 5 min. After SDS-polyacrylamide gel electrophoresis (4%–20% gradient), proteins were transferred onto PVDF membranes. The membranes were blocked in blocking buffer (1% w/v BSA, 1% w/v non-fat powdered milk in phosphate buffered saline with 0.01% v/v Tween-20 (PBST) buffer) before incubation with primary antibodies in blocking buffer overnight at 4°C. Membranes were washed in PBST buffer and incubated with anti-mouse or anti-rabbit IgG HRP-linked antibodies in blocking buffer. Protein levels were detected using Amersham ECL Prime detection reagent and an Amersham Imager 600 instrument (GE Healthcare).

Isolated mouse T cells were obtained as described above. One million cells were resuspended in 30 μl of 10% FBS medium or 10% delipidated FBS medium with CD3/CD28 activation beads in a bead-to-cell ratio of 1∶1. After 2 min of incubation, cells were mixed by pipetting up and down 5 times in RIPA buffer (Sigma-Aldrich) containing complete, Mini protease inhibitor cocktail tablet (Roche Diagnostics) Proteins were extracted by freeze and thaw three times and then centrifuged at 13793 g for 20 min at 4°C. Protein concentrations were determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Ten μg of protein were loaded on a 12% SDS-Page gel and then transferred to nitrocellulose membrane. The membrane was blocked with 5% BSA and then probed with anti phospho-Zap-70, anti Zap-70 or anti α-tubulin (Cell Signaling) as primary antibody and ECL HRP labelled anti rabbit IgG (GE Healthcare) as secondary antibody. Amersham ECL Prime detection reagent (GE Healthcare) was used to visualize protein bands.