Horseradish peroxidase conjugated anti mouse igg antibody

Binding activities of anti-SORT1 mAbs were tested using ELISA. Briefly, human or mouse SORT1 were immobilized on a 96-well plate and anti-SORT1 mAb was reacted to the plate-bound SORT1. SORT1-reactive mAb was detected using a horseradish peroxidase-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) and SureBlue/TMB peroxidase substrate. The reaction was stopped by adding H₂SO₄, and OD₄₅₀ was measured using a SpectraMax 340PC384 plate reader (Molecular Devices).

On day 7, the samples were lysed
at 4 °C with lysis buffer for 1 h and stored at −80 °C.
Before Western blotting, we centrifuged the lysates at 4 °C for
10 min at 13,000 rpm.
We evaluated the protein content with
a bicinchoninic acid protein assay kit (PierceTM; Thermo Fisher Scientific)
and loaded 20 μg of protein in an 8% sodium dodecyl sulfate
polyacrylamide gel electrophoresis system.
We transferred the
electrophoresis gel to a nitrocellulose membrane
(BioRad Laboratories). We incubated the membrane with albumin monoclonal
primary antibody (Cederlane, 1:1000) or p-glycoprotein
(p-gp) monoclonal antibody (Thermofisher, 1:200)
overnight at 4 °C with horseradish peroxidase-conjugated antimouse
IgG antibody (Jackson ImmunoResearch, 1:15,000) and then used enhanced
chemiluminescence as the detection system (Millipore). We developed
the immunoreactive bands with a Firereader V10 PLUS 26M Imaging system
(Uvitec Ltd.) and quantified by ImageJ software. The obtained values
were then normalized on the GAPDH signal coming from the same samples.

Thapsigargin (TG), melatonin, rabbit polyclonal anti-β-actin antibody (catalog number A2066, epitope: amino acids 365–375 of human β-actin), bovine serum albumin (BSA), hydrocortisone (catalog number: H0888), insulin (catalog number: I9278), epidermal growth factor (catalog number: E9644), OAG and cholera toxin (catalog number: C8052) were from Sigma. Fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes. Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone. Rabbit polyclonal antiubiquitin antibody (catalog number: ab19247) was from Abcam. DharmaFECT kb transfection reagent was from Cultek. Mouse monoclonal anti-PMCA antibody (Clone 5F10, epitope: amino acids 724–783 of human PMCA) and Live/Dead viability/cytotoxicity kit were from Thermo Fisher. Horseradish-peroxidase-conjugated anti-mouse IgG antibody and anti-rabbit IgG antibody were from Jackson ImmunoResearch Europe Ltd. RNA control vector was from Origene. Enhanced chemiluminescence detection reagents were from Pierce. Bromodeoxyuridine (BrdU) cell proliferation assay kit was from BioVision. SAR7334 was from Quimigen. hTRPC6-YFP was a gift from Craig Montell (Addgene plasmid#21084; http://n2t.net/addgene:21084; RRID:Addgene_21084). All other reagents were of analytical grade.