Anti rabbit igg conjugated with horseradish peroxidase

Immunization of a rabbit with the purified recombinant TbGMPR as an antigen, and preparation of the antiserum were performed by Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). Whole IgG was fractionated from the antiserum by ammonium sulfate precipitation followed by Ab-Rapid SPiN EX column chromatography (ProteNova Co. Ltd., Kagawa, Japan). Crude lysates of T. brucei were prepared in RIPA buffer and subsequent centrifugation. The lysates were separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane (Bio-Rad). The membrane was then incubated overnight at 4°C with 5 μg/ml of the anti-TbGMPR polyclonal IgG in Tris-buffered saline with 0.05% Tween-20 (TBST) containing 5% skim milk (Wako). After having been washed with TBST, the blots were incubated with anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technologies, Danvers, MA). The immunoreactivities were visualized by using ImmunoStar Zeta (Wako) and LAS4000 imaging device (GE Healthcare Japan).

Aliquots of samples were suspended in 4× Laemmli buffer (Bio-Rad) with 2-mercaptoethanol (Sigma) and incubated for 5 min at 98°C. The protein samples (10 μL each) were run on 4 to 20% Mini-Protean TGX stain-free precast gels (Bio-Rad) in 1× TGX buffer and then transferred to a nitrocellulose membrane using a Trans-Blot Turbo transfer system (Bio-Rad). Subsequently, the membranes were blocked using blocking buffer consisting of 5% skim milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST) for 2 h at 4°C with agitation. The membranes were then incubated in PBST containing 1% skim milk with primary antibodies, polyclonal rabbit antiserum raised against ExbB and TolC (kindly given by Philippe Delepelaire), GFP (Invitrogen; A6455; Thermo Fisher Scientific, Indianapolis, IN, USA), and mCherry (Invitrogen; PA5-34974) at 1:20,000 overnight at 4°C with agitation. The membranes were washed in PBST and incubated in PBST containing 1% skim milk with a secondary antibody, anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling; 7074S), at 1:10,000 for 2 h at 25°C with agitation. After washing the excess secondary antibody, specific bands were visualized using the enhanced chemiluminescence (ECL) prime detection method (GE Healthcare) and imaged with an imaging system, iBright CL1500 (Invitrogen).

Protein lysates (40 µg per lane) were loaded on a 4–15% SDS-PAGE gel (Bio-Rad), separated in 1× Tris-Glycine Buffer (Bio-Rad), and transferred to PVDF membranes via a wet electro-blotting system (Bio-Rad), all according to the manufacturer’s instructions^{52 (link)}. PVDF membranes were blocked for 1 h in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T, Bio-Rad), then incubated overnight at 4 °C with antibodies listed in Supplementary Table 2 in 5% bovine serum albumin (BSA) in TBS-T. Blots were then probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1:3000, Cell Signaling Technology) in 5% BSA in TBS-T for 1 h at room temperature. Signal was detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA), and blot images were collected with a Bio-Rad ChemiDoc imaging system.

RPMI-1640 medium, fetal bovine serum and other culture reagents were obtained from Gibco Life Technologies (Grand Island, NY, USA). The cell culture plates were purchased from Nalge Nunc International (Roskilde, Denmark). Collagenase type XI was purchased from Sigma (St. Louis, MO, USA). The anti-3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-rabbit IgG conjugated with horseradish peroxidase was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

After extraction using RIPA buffer, 10 μg of total proteins were separated by 8–12% SDS PAGE and then transferred onto nitrocellulose membrane. After 1h in blocking buffer (5% skim milk in TBS-Tween (TBST)), membranes were washed three times with TBST and incubated overnight at 4°C with primary antibodies against mouse FGFR1, FGFR3, FGFR4 (Abbiotec, USA) used at a dilution of 1/200 or against β-actin (Sigma-Aldrich, France) used at 1/8000. After three 5min-washing steps with TBST, blot were incubated for 1h at room temperature with anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technology, USA) at 1/4000 dilution in blocking buffer. After three 5min-washes in TBST, signal was detected by chemiluminescence (ECL Plus Western blotting detection reagent, Biorad, France).