Anti rabbit peroxidase conjugated secondary antibody

For western blot analysis, equal amounts of proteins for each sample were separated by SDS-PAGE and then electroblotted onto PVDF membranes (0.45 μM, Millipore, USA), and immunoblotted overnight at 4 °C with primary antibodies^{21 (link)}. The membranes were then incubated with anti-rabbit peroxidase-conjugated secondary antibody (1:5000, Jackson Labs), visualized with Clarity Western ECL Blotting Substrates (Bio-Rad Laboratories, USA)^{22 (link)}. The relative quantitative PCRs (qPCR) were carried out with SYBR Premix Ex Taq kit (TaKaRa Bio, Dalian, China) on a QuantStudio6 Flex thermocycler (Applied Biosystems, Carlsbad, CA, USA)^{2 (link)}, and the primers used in PCR reaction are presented in Table S2.

Soluble proteins from tissues were obtained by overnight shaking (600 RPM) in 0.5 M NaCl, 10 mM Tris Base pH 7.5, 1× protease inhibitor buffer at 4 °C. After centrifugation at 13,000 RPM for 1 min, the supernatants (soluble proteins) were collected and quantified. Samples (10 γg) were separated in NuPAGE™ 4–12% Bis–Tris Midi Protein Gels (Life technologies, Thermo Fisher Scientific, Waltham, MA, USA) by SDS-PAGE with MOPS running buffer (Life technologies) and electroblotted onto nitrocellulose (GE, Healthcare, Little Chalfont, UK) membranes. The blots were incubated with primary and secondary antibodies. The antibodies were revealed using Luminata™ Western HRP Chemiluminescence Substrates (Millipore Corporation, Billerica, MA 01821 U.S.A). To control for equal protein loading and transfer, the membranes were stained with Ponceau S solution (Sigma, St. Louis, MO, USA). The following antibody was used: anti-COL3A1 (Santa Cruz Biotechnology, Dallas, Texas, USA, sc-8781). The secondary anti-rabbit peroxidase-conjugated antibody was from Jackson ImmunoResearch (Cambridge House, UK). The chemiluminescent blots were imaged with the ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Dilutions 1:10 of the serum samples were separated in 12% gels (Life technologies) by SDS-PAGE with MES running buffer (Life technologies) and electroblotted onto nitrocellulose (GE, Healthcare, Little Chalfont, UK) membranes. The blots were incubated with primary and secondary antibodies. The antibodies were revealed using ECL (Millipore). To control for equal protein loading and transfer, the membranes were stained with Ponceau S solution (Sigma). The following antibodies were used: anti-C-VTN (Abcam, Cambridge, UK, ab113700), anti-N-VTN (Santa Cruz Biotechnology, Dallas, Texas, USA, sc-15332); the secondary anti-rabbit peroxidase-conjugated antibody was from Jackson ImmunoResearch. The chemiluminescent blots were imaged with the ChemiDoc^TM Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and vitronectin band densities were quantified by ImageLab software version 5.1.2 (Bio-Rad). Total protein lane densities by Ponceau S staining were used for normalization.

Pituitaries (n = 6 per group) and inguinal lymph nodes were homogenized in lysis buffer (Abcam, Cambridge, MA, USA) by mortar and pestle in liquid nitrogen. Lysates were centrifuged to remove debris and quantified by a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Cleared lysates (60 μg/lane) were separated by a reducing SDS–PAGE and transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin in PBST (PBS containing 0.05% Tween-20 (VWR-Amresco, Radnor, PA, USA)), the membranes were incubated with an anti-IRAK1 antibody (1:1000, Cell Signaling, Danvers, MA, USA), followed by a peroxidase-conjugated anti-rabbit secondary antibody (1:20,000, Jackson ImmunoResearch, West Grove, PA, USA). The blots were developed by adding a chemiluminescent substrate (Thermo Scientific), and chemiluminescence images were recorded by a CCD imaging system (Hansor, Taichung, Taiwan). The images of IRAK1 and β-actin blots on the same membrane were quantified with the Image J software (version 13.06 for Mac OS X, 64 bit, free software, National Institutes of Health, Bethesda, MD, USA (accessed on 25 August 2021)). Signal intensities of IRAK1 were divided by the intensities of β-actin to obtain normalized IRAK1 expression.