Hrp labeled anti rabbit igg

CSF and serum samples were dissolved in Laemmli buffer without 2-mercaptoethanol, boiled for 3 min, and loaded on SDS-polyacrylamide gels (194–1502, FUJIFILM Wako) [27 (link)]. After SDS-PAGE, protein bands were visualized with a Silver Stain II kit (FUJIFILM Wako). For immunoblotting, proteins separated on gels were transferred to nitrocellulose membranes. The membranes were blocked in 3% skim milk, and incubated sequentially with the following combinations of primary and secondary antibodies: anti-Tf antibody (Bethyl Laboratories, Montgomery, TX, USA) and horseradish peroxidase (HRP)-labeled anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA); anti-PDGS antibody (PA1-46023, Thermo Fisher Scientific, Waltham, MA, USA) and HRP-labeled anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA); anti-TTR antibody (ab9015, Abcam, Cambridge, CB2 0AX, UK) and HRP-labeled anti-sheep IgG antibody (#31480, BiotechnologyThermo Fisher Scientific). The protein bands were visualized with a SuperSignal West Dura Chemiluminescence Substrate Kit (Pierce Biotechnology, Rockford, IL, USA). Purified standards of GlcNAc and Man-Tf mixture were purified from CSF as described previously [15 (link)]. His-tagged TTR (89-7754-27) and L-PGDS (E-PKSH030653.10) were purchased from Elabscience Biotechnology Inc., (Houston, TX, USA) and Biomol (Hamburg, Germany)

Protein samples from rat liver or miR-322 overexpressing-cells were extracted using complete lysis-M (Roche, Mannheim, Germany). The protein concentrations of lysate samples were determined using the Pierce 660 nm Protein assay (Thermo Scientific, Rockford, IL). Each 40 µg protein was electrophoresed on a 4–15% gradient Criterion TGX gel and transferred to a nitrocellulose membrane. The transfer membranes were blocked with 4% skim milk and then incubated with an anti-GHR antibody (1: 200, ab134078, abcam, Cambridge UK) for 1 hour at room temperature followed by an additional overnight incubation at 4 °C. The transfer membranes were washed with TBS-T, and further incubated with HRP-labeled anti-rabbit IgG (1: 2000, Jackson Immunoresearch, West Grove, PA) for 1 hour at room temperature. The signals were detected using SuperSignal West Dura extended duration substrate (Thermo Scientific). The membranes after detection were stripped of the antibody using Restore plus western blot stripping buffer (Thermo Scientific). Then, signals were detected again using THE^TM [HRP] -labeled beta actin antibody (1:1,000 A00730-40, GeneScript, Pitcataway, NJ). The expression levels of GHR were quantified by correcting the GHR signal with the ß-actin signal.