Horseradish peroxidase conjugated anti mouse igg

Conditioned medium was concentrated 12.5-fold with Nanosep microconcentrators (3 kDa MW cut-off; Pall Life Sciences, Port Washington, NY, USA) before separation of reduced samples by 10% SDS-PAGE and Western blot. The primary antibody (mAb-5/6), targeted to a region near the C-terminus of AMH (used at a 1:5000 dilution), was from Abcam (Cambridge, UK). The secondary antibody (diluted 1:10 000) was horseradish peroxidase-conjugated anti-mouse IgG (GE Healthcare, Buckinghamshire, UK), with detection of immunoreactive proteins using Lumi-light chemiluminescence reagents (Roche, Basel, Switzerland) and a ChemiDoc^™ MP system (Bio-Rad, Hercules, CA, USA) with Image Lab^™ software (Bio-Rad). A horseradish peroxidase-conjugated anti-GAPDH monoclonal antibody (diluted 1:10 000) was used as an internal control (Cell Signaling Technology, Danvers, MA, USA). The AMH prodomain was detected with a mouse-derived monoclonal His-tag antibody (R&D Systems, Minneapolis, MN, USA; used at a 1:1000 dilution), targeted to the 6×His epitope-tag located at the N-terminus of the prodomain.

For western blotting, 1 μg of each of the recombinant proteins was separated by SDS-PAGE and transferred to Hybond-P membrane (GE Healthcare, QC, Canada). The membrane was blocked with 5% skim milk dissolved in phosphate buffered saline (PBS) containing 0.05% tween-20 (PBS-T) for 2 hr at room temperature. Then, it was incubated overnight at 4°C with mice serum that was collected four weeks after the last immunization with the respective antigen. After washing three times with PBS-T, the membrane was incubated with a horseradish peroxidase-conjugated anti-mouse IgG (GE Healthcare, QC, Canada) for 45 min at room temperature (RT) followed by three washing steps. Immunoreactivity was detected by chemiluminescence using ECL reagents following the manufacturer's instructions (GE Healthcare, QC, Canada).

The rabbit polyclonal antibody (pAb) against β-catenin (ab6302) and the mouse monoclonal antibody (mAb) against DSG3 (3G133) were purchased from Abcam (Cambridge, UK). The mouse inhibitory mAb against E-cadherin (67A4) and the mouse mAb against cyclin E (HE12) were from Merck Millipore (Billerica, MA). The rabbit pAb against Ser127-phosphorylated YAP (#4911) and the rabbit mAb against E-cadherin (24E10) were from Cell Signaling Technology (Danvers, MA). The rabbit pAb against YAP1 (NB110-58358) was from Novus Biologicals (Littleton, CO). The mouse mAbs against β-actin (AC-15) and vinculin (hVIN-1), and the rabbit pAb against α-catenin (C2081) were from Sigma Chemical (St. Louis, MO). Mouse IgG1 for the isotype control (2E12) was from Medical & Biological Laboratories (Nagoya, Japan). Alexa Fluor 488-goat anti-rabbit IgG, Alexa Fluor 488-goat anti-mouse IgG and Alexa Fluor 546-goat anti-mouse IgG antibodies, and Alexa Fluor 546-phalloidin were from Life Technologies. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were from GE Healthcare (Little Chalfont, UK) and Life Technologies, respectively. The recombinant E-cadherin-Fc chimeric protein, Y-27632, iCRT3 and Verteporfin were from Sigma Chemical. Blebbistatin was from Toronto Research Chemicals (North York, Canada). The RhoA activator CN03 was from Cytoskeleton (Denver, CO).

Western blotting analysis was done as previously described [23 (link)]. Briefly, 175 + 20 mg of frozen mouse brain tissue were homogenized in 5% glucose distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad). To detect brain PrP^res 100 μL of 10% (wt/vol) brain homogenate were subjected to digestion with 40 μg/mL of PK in buffer 5% sarkosyl, 5% Triton X100, 1 M Urea, and 16 mM Tris–HCl (pH 9.6) at 60 °C for 15 min. Digested samples were loaded samples into 12% Bis-Tris Gel (Criterion XT; Bio-Rad). After electrophoretic transference of proteins onto polyvinylidene fluoride membranes (Millipore) and blocking overnight with 2% bovine serum albumin blocking buffer, membranes were incubated with 12B2 [24 (link)] and Sha31 [25 (link)] mAb at a concentration of 1 μg/mL. 12B2 recognizes the 89-WGQGG-93 epitope of the human-PrP^C sequence while Sha31 recognizes the 145-WEDRYYRE-152 epitope of the human-PrP^C sequence. Immunocomplexes were detected by 1 h membrane incubation with horseradish peroxidase conjugated antimouse IgG (GE Healthcare Amersham Biosciences) and development with enhanced chemiluminescence in ECL Select (GE Healthcare Amersham Biosciences). Images were captured using the ChemiDoc WRS+ System and processed them by using Image Lab 5.2.1 software (both Bio-Rad).