Secondary antibodies conjugated with horseradish peroxidase

Lysates from sFBs were prepared using RIPA buffer (25 mM Tris–HCl [pH 7.6], 150 mM sodium chloride, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) containing protease inhibitors (Roche, Basel, Switzerland) according to the standard method [13 (link)], followed by centrifugation at 14,000×g for 15 min at 4 °C, and the concentration of each sample using a Bio-Rad protein assay kit (Bio-Rad, Tokyo, Japan) with bovine serum albumin as a standard. The sample was electrophoresed in 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane and incubated with primary antibodies: FGF7 (1:1000; abcam, Tokyo, Japan), FGF9 (1:1000; abcam, Tokyo, Japan), and GAPDH (1:5000; Sigma-Aldrich, Tokyo, Japan). After washed by TBST (20 mM Tris–HCl, 150 mM NaCl, and 0.02% Tween-20, pH 7.4), the blots were incubated with secondary antibodies conjugated with horseradish peroxidase (1:4000, anti-rabbit; GE Healthcare, Tokyo, Japan) for 1 h at room temperature. The bands were detected using ECL-Plus Substrate (GE Healthcare, IL, USA) and exposed to Hyperfilm (GE Healthcare, IL, USA). The data was obtained from an experiment using samples from a single donor.

The antibodies for caspases-3, caspase-8, STAT3, pTyr705-STAT3, pSer727-STAT3, pSer70-bcl2, Bid, Bad, Akt, p38MAPK, pThr180/Tyr182-p38MAPK, cytochrome c, were from Cell Signaling Technology (Beverly, MA, USA). Antibodies for Bax and p21^CIP1 were from MBL (Nagoya, Japan). Antibodies for β-actin, Rb, Mcl-1, bcl-2, bcl-xL, p-gp130, JAK2, Cyclin D2, cyclin E, CDK2, CDK4, PKCα, PP2A/Aα, PP2A/B56α, PP2A/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for p-JAK2 or p27^kip1 were from Upstate (Waltham, MA, USA) or BD Transduction Laboratories, respectively. Anti-ERK1/2 or p-ERK1/2 antibody was from Sigma. Secondary antibodies conjugated with horseradish peroxidase were obtained from GE Healthcare (Tokyo, Japan).

Eluates were supplemented with RNase buffer to a final concentration 20 mM Tris–HCl pH 7.5, 150 mM NaCl and 200 U ml⁻¹ of RNases A and T1. After 30 min incubation at 37 °C an aliquot of proteins was supplemented with LiDS loading buffer (Thermo Fischer Scientific) containing 20 mM DTT, separated on 4–12% polyacrylamide gels (NuPage, Thermo Fischer Scientific) and either stained with silver using Pierce Silver Stain Kit (Thermo Fischer Scientific) or electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes.
PVDF membranes were blocked in phosphate buffered saline supplemented with 0.1% Tween-20 (PBST) and containing 100 g L⁻¹ milk powder. Membranes were then hybridized with antibodies diluted in PBST-milk that are listed in Supplementary Table 2. Membranes were washed with PBST, hybridized with secondary antibodies conjugated with horseradish peroxidase (GE Healthcare) and developed using Western Lightning Plus ECL kit (PerkinElmer). Full-size images of the most important immunoblots are available in the Supplementary Fig. 1.