Amersham ecltm western blotting detection reagents

SDS-PAGE and Western blotting in the analyses of amyloid fibril and soluble Aß were performed as previously reported [28 (link)]. Briefly, the samples dissolved in SDS-PAGE sample buffer were subjected to PAGE using 4–20% gradient gel and Tris/tricine buffer (both Cosmo Bio, Tokyo, Japan). The separated proteins were electrotransferred onto a nictocellulose membrane, and the membrane was incubated in 5% skim milk in PBS-T (PBS containing 0.05% Tween 20) for 1 hr at RT. Aß was detected using the anti-Aß anibody 6E10 (Covance, Princeton, NJ), an HRP-conjugated secondary antibody, and Amersham ECL^TM Western Blotting Detection reagents (GE Healthcare, UK).

Jejunal IEC from three-month-old wild-type and villin-Cre Hdac1^−/−; Hdac2^−/− mice were enriched by EDTA treatment. IEC were lysed in 1 X Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the Pierce BCA Protein Assay kit (Therrmo Fisher Scientific). Amounts of 15 µg of total proteins were separated on 4–12% SDS-polyacrylamide gels, and transferred on PVDF membranes (Roche Molecular Biochemicals). Membranes were incubated 1 h at room temperature or overnight at 4 °C with primary antibodies including rabbit anti-phospho-Stat3 (#9145, Cell Signaling), rabbit anti-Stat3 (#12640, Cell Signaling), rabbit anti-phospho-p38 (#4511, Cell Signaling), rabbit anti-claudin 3 (341700, Invitrogen), rabbit anti-cleaved Notch (#4147, Cell Signaling), rabbit anti-phospho-S6 (#4858, Cell Signaling) and GAPDH (#2118, Cell Signaling). Primary antibodies were recognized with secondary goat anti-rabbit antibodies (Life Technologies) before immune complex detection with Amersham ECL^TM Western blotting detection reagents (GE Healthcare, Mississauga, ON, Canada).

Five-day wild-type, Hdac1- or Hdac2-deleted enteroid cultures were used, unless otherwise stated. Enteroids were recovered and directly lysed in 1 X Laemmli buffer 1 (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol) supplemented with protease and phosphatase inhibitors. Whole enteroid protein content was measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Fifteen µg of total protein extracts were added on 4–12% SDS-polyacrylamide gels, and transferred on PVDF membranes (Roche Molecular Biochemicals). Western blotting was performed as described previously^{79 (link)}. Membranes were incubated 1 h at room temperature or overnight at 4 °C with the following primary antibodies: rabbit anti-HDAC1, rabbit anti-HDAC2 (Abcam); rabbit anti-HMGCS2, rabbit anti-phosphoSTAT3, rabbit anti-STAT3 (Cell Signaling); mouse anti-ACTIN (EMD Millipore). Secondary antibodies included goat anti-mouse and goat anti-rabbit (Invitrogen). Immune complexes were revealed with Amersham ECL^TM Western blotting detection reagents (GE Healthcare) (n = 3). Additional information about antibodies is included in Supplementary Table S8.

Whole cell lysates were prepared in RIPA buffer (Sigma) and quantified using Bradford Protein Assay (Bio-Rad). Twenty micrograms of lysates were resolved on Bolt^® 4–12% Bis-Tris Plus gels using Bolt^TM MOPS SDS Running buffer and transferred to a Hybond C super nitrocellulose membrane (GE Healthcare). After blocking to prevent non-specific binding in 5% milk in PBST for 1 h at room temperature, membranes were incubated with the specific primary antibodies overnight at 4 °C. The following primary antibodies were used: E-cadherin (Takara Bio Inc., M106, clone HECD-1, 1:1000 dilution), Occludin (Cell Signaling, #5446, 1:1000 dilution), Vimentin (Cell Signaling, #5741, 1:3000 dilution), ZEB1 (Santa Cruz, sc-25388, 1:500 dilution), SNAI1 (Santa Cruz, sc-28199, 1:500 dilution), LIN28B (Cell Signaling, #4196, 1:1000 dilution), LIN28A (Cell Signaling, #3978, 1:1000 dilution), SMAD2/3 (Cell Signaling, #8685, 1:1000 dilution), β-actin (Abcam, ab8227, 1:200,000 dilution), GAPDH (Santa Cruz, sc-137179, 1:10,000 dilution). Following incubation with the specific HRP-conjugated antibody (Dako, #P0447 or #P0448, 1:2,500 dilution), chemiluminescence signal was detected using Amersham^TM ECL^TM Western blotting detection reagents (GE Healthcare) and Amersham Hyperfilm^TM ECL (GE Healthcare). Uncropped scans of the most important blots are shown in Supplementary Fig. 13.

Cells were lysed with RIPA buffer (Thermo Fisher Scientific, USA) and denatured at 95 °C before being resolved on 8% or 10% SDS-PAGE. Separated proteins were then transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA) and blocked with 5% milk. The membrane was incubated with primary antibody overnight at 4 °C, followed by secondary antibody incubation for 1 h at room temperature. Chemiluminescent signal was detected with ChemiDoc^TM MP imaging System (Bio-Rad, USA) using Amersham^TMECL^TM Western Blotting Detection Reagents (GE Healthcare, UK). Image processing and band densitometry were done with Image Lab^TM 6.0.1 software (Bio-Rad, USA). β-tubulin was used as loading control. All antibodies used were listed in Supplementary Table 1. Un-cropped western blots accompanied by size markers were presented in Supplementary Fig. 8.