Hrp conjugated donkey anti goat igg

Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and LPCAT4 siRNA (ON-TARGET plus mouse MBOAT2: L-063482-01) were purchased from GE Healthcare (Little Chalfont, UK). Anti-Col2 polyclonal antibody, anti-Col10 polyclonal antibody, control siRNA (SC: 37007), and HRP-conjugated donkey anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin monoclonal antibody, bovine serum albumin (BSA), and 0.25% trypsin-EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Commercial IgA1 or purified IgA1 from IgAN or non-IgAN patient serum or artificial IgA1-IgG immune complexs was resolved in 12% SDS-PAGE gel and transferred to PVDF membrane (Minipore, USA). After 1-hour’s blocking with 2.5% bovine serum albumin, HRP-conjugated mouse anti-human IgA1 monoclonal antibody (Fc fragment specific, 1:1000, Abcam, USA) or HRP-conjugated donkey anti-goat IgG (1:1000, Santa Cruze, USA) was employed to recognize the total IgA1 at 4 °C overnight. The membrane was then washed with TBST to remove unbound antibody followed by signal development with Immobilon Western Chemiluminescent HRP Substrate (Minipore, USA). For detection of galactose-deficient IgA1 by western blot, the blot was firstly treated with 15 mU/ml neuraminidase for 6 hours at 37 °C after membrane transfer. After washing with TBST, biotinylated HAA (1:200, Sigma, USA) was used to bind the GalNAc-exposed IgA1 overnight at 4 °C and HRP-conjugated avidin (1:5000, Sigma, USA) was introduced to recognize biotin for 1 hours at 37 °C. Signal development was the same as above. Gray intensity of western blot signal band was calculated with Image J 1.47V software (NIH, USA).

Cell lysates were collected in 1×RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA) supplemented with Complete Mini, EDTA-free protease inhibitor (Roche Applied Science, Indianapolis, IN). Protein concentration was assayed by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Proteins were resolved on 4–20% SDS-PAGE gel and transferred to PVDF membrane using Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad, Hercules, CA). Primary antibodies used for western blot analysis were rabbit monoclonal RIG-I (D14G6), cleaved CASP3 (Asp175) (5A1E), cleaved PARP (Asp214) (D64E10) XP (Cell Signaling technology, Inc., Danvers, MA), IRF3 antibody [EP2419Y] (Abcam, Cambridge, MA), and goat polyclonal anti-actin (I-19) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibodies used were HRP-linked goat anti-rabbit IgG (Cell Signaling technology, Inc., Danvers, MA) and HRP-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immune complexes were visualized with Amersham ECL Western Blotting Detection Reagents and exposed to X-ray films (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) according to the manufacturer’s instruction. The protein level was quantitated by densitometry measurement using AlphaEaseFC software (Alpha Innotech, Miami, FL).

Total protein was extracted from MC3T3-E1 cells on the Ti discs using a NP-40 lysis buffer. After electrophoresis and transferring protein, the membrane was blotted with the primary antibodies for 16 h at 4°C, such as 1:2000 of anti-rabbit Runx-2 (SantaCruz Biotechnology Inc., Dallas, TX, USA), 1:2000 of anti-goat OPG (SantaCruz Biotechnology Inc.), 1:1000 of anti-mouse RANKL (Novus Biological Inc., Centennial, CO, USA) and 1:2,500 of anti-mouse β-actin (Santa Cruz Biotechnology Inc.). After washing, the membrane was blotted with 1:5000 of either horseradish peroxidase (HRP)-conjugated goat anti-rabbit or mouse-IgG (Enzo Life Sciences Inc., New York, NY, USA) and HRP- conjugated donkey anti-goat-IgG (SantaCruz Biotechnology Inc.). The developing was performed using X-ray film (Fuji Film Co.) after detection using an ECL solution (Merck Millipore, Burlington, MA, USA). The density of the expressed bands was measured using Science Lab Image Gauge (Fuji Film Co.).