Mouse anti actin

Samples were subjected to SDS-PAGE and transferred to nitrocellulose or PVDF membranes. Blots were blocked for 1 h in 5% (w/v) skim milk-PBST (0.1% Tween 20 in PBS) and then incubated with the primary antibody diluted in 5% skim milk-PBST for 1 h at room temperature. The optimal dilution for each antibody was calibrated individually. The secondary antibodies were diluted in 5% skim milk-PBST, incubated with the blots for 1 h at room temperature and detected with ECL reagents. The following commercial antibodies were used: mouse anti-DnaK (Abcam), mouse anti-JNK (BD Pharmingen), and mouse anti-actin (MPBio). Antibodies directed against T3SS components were a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada) and included mouse anti-EspA, mouse anti-EspB, rat anti-EscJ, mouse anti-Tir, and rabbit anti-SigD. Secondary antibodies were HRP-goat anti-mouse (Abcam) and HRP-goat anti-rat (Jackson ImmunoResearch).

Samples were subjected to SDS-PAGE and transferred to nitrocellulose (pore size: 0.45 μm; Amersham Protran) or PVDF (pore size: 0.45 μm; Amersham Hybond) membranes. The blots were then blocked for 1 h with 5% (w/v) skim milk-PBST (0.1% Tween in phosphate-buffered saline), incubated with the primary antibody (diluted in 5% skim milk-PBST, for 1 h, at room temperature, unless otherwise indicated), washed, and then incubated with the secondary antibody (diluted in 5% skim milk-PBST, for 1 h, at room temperature). Chemiluminescence was detected with EZ-ECL reagents (Biological Industries). The following primary antibodies were used: mouse anti-HA (Abcam Inc.), diluted 1:1,000; mouse anti-HA.11 (Covance), diluted 1:1,000; mouse anti-V5 (Invitrogen), diluted 1:1,000; mouse anti His (Pierce), diluted 1:2000; mouse anti-JNK (BD Pharmingen), diluted 1:1,000 in Tris-buffered saline (TBS); and mouse anti-actin (MPBio), diluted 1:10,000. Antibodies directed against T3SS components included mouse anti-EspA, mouse anti-EspB and mouse anti-Tir, all a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada). Horseradish peroxidase-conjugated (HRP)-goat anti-mouse (Abcam Inc.), diluted 1:10,000, was used as the secondary antibody. Representative western blots of at least three independent experiments are presented in the results section.

Whole cellular protein extracts were prepared in 95°C Laemmli sample buffer and mechanically sheared by pressing few times through syringes (26 G). Protein concentration was determined using a Pierce™ 660 nm Protein Assay (Thermo Scientific). A total of 5 µg protein per extract was separated on denaturing 10–20% gradient SDS-PAGE gels. Proteins were transferred on PVDF transfer membranes (0.45 µm, Perkin Elmer). Membranes were probed with the following primary antibodies: anti-calretinin (Sigma, HPA007306), mouse anti-actin (#69100) from MP Biomedicals, N-Cadherin (BD Biosciences, 610920), YAP (Cell Signaling 4912), Mesothelin (Rockland Inc. 200-301-A88), GATA4 (C-20) (Santa Cruz sc-1237), p62 (Progen GP62-C), LC3B (Cell Signaling 2775S), p53 (DO-1) (Santa Cruz sc-126), Thy1 (H-110) (Santa Cruz sc-9163), and γ-H2AX (Millipore 05-636). Membranes were then incubated with the secondary antibody rabbit anti-mouse IgG-HRP (A-5420) from Antell, and goat anti-rabbit IgG-HRP (#7074) from Cell Signaling. The signals were detected by enhanced chemiluminescence (ECL™ Western Blotting Reagents, GE Healthcare) and detected on photosensitive film (Super RX Fuji x-Ray Film, Fujifilm). Proteins were quantitated with densitometry using Image J software (Version 1.42q, USA).

Protein extraction, SDS-PAGE separation, and visualization were performed as described [73 (link)], except for the detection step (Immobilon Forte, Millipore WBLUF0500) and band intensity quantification (ImageJ software). Western blot analysis of media was performed similarly, without a lysis step (media were directly diluted with sample buffer X5 and denaturized by heating).
Antibodies: mouse anti-Actin (MP biomedical 69100, 1:10,000), mouse anti-β-catenin (BD 610154, 1:2000), rabbit anti-c-Myc (Santa Cruz 788, 1:500), rabbit anti-Frizzled (Santa Cruz Biotechnology 9169, 1:500), mouse anti-GSK3 (Santa Cruz Biotechnology 7291, 1:500), rabbit anti-LEF1 (Abcam 137872, 1:1000), mouse anti-LRP5 (Abcam 129357, 1:500), rabbit anti-LRP6 (Abcam 134146, 1:1000), mouse anti-TCF4 (Millipore 05–511, 1:1000), rabbit anti-Wnt-3a (Cell Signaling 2721, 1:3000), Goat anti-mouse HRP (Jackson ImmunoResearch 115–035-003, 1:10000), Goat anti-rabbit HRP (Jackson ImmunoResearch 111–035-144, 1:10000).

MCF-7 and MDA-MB-231 cells were lyzed in MPER (Thermo Scientific, Bleiswijk, The Netherlands) and diluted 1:1 with SDS sample buffer (4% SDS, 20% glycerol, 0.5 mol/l Tris-HCl (pH 6.8), 0.002% bromophenol blue). Lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight at 4 °C and probed with the following antibodies: rabbit-anti-AKT, rabbit-anti-pAKT (Thr308), rabbit-anti-S6, rabbit-anti-pS6, rabbit-anti-4EBP1 (all Cell Signaling Technologies, Leiden, The Netherlands) in a 1:1000 dilution or anti-HIF1α (BD Biosciences, Breda, The Netherlands) and mouse-anti-actin (MP Biomedicals, Santa Ana, USA) in a 1:10,000 dilution. Primary antibodies were stained using HRP-coupled goat anti-rabbit or rabbit anti-mouse IgG and developed with Lumi-Light (Roche, Almere, The Netherlands). Images were captured with the ChemiDoc MP imaging system (Bio-Rad, Veenendaal, The Netherlands) and Image Lab Software.